Influence of Antioxidant Sericin in Tris Extender on Oxidative Markers during Cryopreservation (–196ºC) of Bovine Semen

2019 ◽  
Vol 15 (02) ◽  
pp. 34-38
Author(s):  
Tapasvi M Patel ◽  
A J Dhami ◽  
D V Chaudhari ◽  
M M Pathan
Keyword(s):  
Superoxide Dismutase ◽  
Sperm Motility ◽  
Seminal Plasma ◽  
Winter Season ◽  
Egg Yolk ◽  
Murrah Buffalo ◽  
Bovine Semen ◽  
Bull Semen ◽  
Oxidative Markers ◽  
The Mean

This investigation was carried out during winter season on the semen of three mature, healthy breeding bulls each of Gir cattle and Murrah buffalo breeds. The aim was to assess the effect of different concentration of antioxidant Sericin (0.0, 0.1, 0.25, 0.50 and 1.0%, w/v) in standard Tris fructose egg yolk glycerol (TFYG) extender on cryopreservability of bovine semen based on sperm motility and oxidative markers in seminal plasma of freshly diluted and cryopreserved semen. The mean sperm motility observed in freshly diluted and frozen-thawed Gir bull semen, irrespective of Sericin levels, were 76.93±0.39 and 43.47 ± 0.58 % and in Murrah bulls 78.20±0.38 and 44.10 ± 0.48 %, respectively. The values of malondialdehyde (MDA, μmol/ml) in seminal plasma of freshly diluted and frozen-thawed semen of Gir bulls, irrespective of Sericin levels, were 21.68±0.38 and 24.99 ± 0.56, and in Murrah bulls 21.49±0.57 and 25.60±0.94, respectively. The corresponding values of superoxide dismutase (SOD, U/ml) were 1.77 ± 0.06 and 1.37 ± 0.05 in Gir and 1.18 ± 0.06 and 0.85 ± 0.04 in Murrah bulls, and those of glutathione peroxidase (GPx, nmol/min/ml) 417.10 ± 12.00 and 349.76 ±11.92 in Gir and 385.71±9.21 and 320.02±9.49 in Murrah bull semen. Sperm motility and activities of all three enzymes differed highly significantly (plessthan00.01) between stages. SOD was significantly (plessthan00.05) lower in buffalo than cattle semen. Inclusion of 0.5% and/or 0.25% Sericin in TFYG extender gave better protection to spermatozoa over other levels against ROS mediated injuries as the MDA production was significantly reduced with increased sperm motility and higher levels of SOD and GPx enzymes in the seminal plasma.

scholarly journals Correlations Amongst Functional and Morphological Attributes and Oxidative Markers of Fresh and Cryopreserved Semen of Gir and Murrah Bulls

2020 ◽  
Vol 15 (03) ◽  
pp. 24-29
Author(s):  
A. J. Dhami ◽  
Tapasvi M Patel ◽  
DV Chaudhari
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Winter Season ◽  
Correlation Coefficients ◽  
Plasma Membrane Integrity ◽  
Murrah Buffalo ◽  
Oxidative Markers

This study was undertaken during the winter season on healthy mature Gir cattle and Murrah buffalo bulls (n=3 each). The semen samples (6 ejaculates/bull, total 36 ejaculates) collected in the morning using artificial vagina were evaluated for routine seminal attributes, including acrosomal and plasma membrane integrity. The samples were then diluted @ 100 million sperm/ml with tris fructose yolk glycerol extender without and with sericin @ 0.1, 0.25, 0.5 and 1.0% (w/v), filled in French mini-straws, and frozen in LN2 using biofreezer as per standard freezing protocol. Straws were thawed in water bath at 37°C for 30 sec and evaluated for post-thaw quality, viz., motility, viability, morphology, acrosome integrity and plasma membrane integrity (HOST). Lipid peroxidation (malondialdehyde - MDA production) and activities of enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) were assessed as oxidative markers in seminal plasma of freshly diluted and frozen-thawed semen samples. Sericn at 0.5% level significantly (p less than 0.01) improved the post-thaw sperm quality with reduced oxidative stress in both the species. The breed-wise correlation coefficients (r) among sperm quality attributes and oxidative markers were studied in fresh and frozen-thawed semen of each species, and also for fresh with frozen-thawed semen. The findings revealed significant interrelationships amongst most of the attributes of fresh as well as post-thawed semen and also of fresh semen attributes with those of cryopreserved semen including oxidative markers in both the species. Sperm motility estimation in fresh, pre-freeze and post-thawed semen was a legitimately good indicator of quality of spermatozoa at various steps of semen processing/freezing, and its fertilizing potential. Thus, the sperm motility, HOS test and either MDA or SOD/GPx activity alone may be used as valuable and practical tools for routine assessment of bovine semen quality considering significant correlations found between them.

95 EFFICACY OF FIVE DIFFERENT SEMEN EXTENDERS FOR THE CRYOPRESERVATION OF BULL SEMEN

2008 ◽  
Vol 20 (1) ◽  
pp. 128 ◽  
Author(s):  
B. Szczesniak-Fabianczyk ◽  
M. Bochenek ◽  
A. T. Palasz ◽  
J. De la Fuente ◽  
Z. Smorag
Keyword(s):  
High Pressure ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Animal Origin ◽  
Membrane Composition ◽  
Bull Semen ◽  
Custom Made ◽  
Significant Difference ◽  
The Mean

Replacement of animal-origin components in extenders used for bull semen freezing is of high importance for individuals involved in cattle breeding. The experiment was designed to compare efficacy of 5 different semen extenders in cryopreservation of bull semen: sodium citrate-based extender containing egg yolk (CT), commercially available Bioxcell� (IMV Technologies, L'Aigle, France), and 3 custom-made homogenized plant lipidsbased, egg yolk-free extenders (Y-1, Y-2, and Lipo) . The objective was to determine whether homogenization procedures of lipids improve the quality of the extender. Lipid homogenates of custom-made extenders were prepared in Tris buffer using a high pressure homogenizer (Nira Saovi, Parma, Italy). Ten (Y-1) or 5 (Y-2) homogenization cycles were applied and then 8% glycerol was added. Lipid liposomes were produced by simultanous high pressure homogenization of lipids and glycerol supplementation (Lipo). Semen was collected from young bulls of 3 different breeds (Simmental, Polish Red, and Holstein; 1 ejaculate/bull). Each ejaculate with at least 70% motility was split into 5 parts and processed further by a standard freezing protocol: semen was diluted at 35�C with each of the 5 extenders to a concentration of 100 � 106 spermatozoa per mL, cooled to 4�C over 5 h, aspirated into 0.25-mL plastic straws, frozen in liquid nitrogen vapor to –140�C, and then plunged into LN2. Straws were thawed in a water bath at 37�C for 20 s. Sperm motility was estimated microscopically immediately after thawing and after 5 h of storage at 22�C. Immediately after thawing, flow cytometry and SYBR-14/PI staining were used for examination of sperm membrane integrity (live/dead assay). A total of 20 000 spermatozoa of each sample were counted. Student's t-test was used to estimate statistical differences between experimental groups. The mean sperm motility after thawing ranged from 45.6% (SD = 13.7) for CT (egg yolk extender) to 57.8% (SD = 7.1) for Lipo. A significant difference (P < 0.05) was observed betweenY-1 (50.0%, SD = 9.7) and Lipo and Bioxcell (56.1%, SD = 8.6). After 5 h of storage at 22�C, the mean motility for all tested bulls ranged from 25.0% (SD = 7.1) for CT to 42.2% (SD = 7.5) for Lipo. Significant differences were observed between Lipo (P < 0.01), Y-2 (P < 0.05) and CT, and between Y-1 and Lipo (P < 0.01). Mean percentage of 'live' spermatozoa with intact membrane after freezing/thawing was 51.85% (SD = 11.49) for Y-1, 45.72% (SD = 9.36) for Y-2, 47.57% (SD = 7.93) for Lipo, 45.47% (SD = 8.35) for Bioxcell, and 49.06 (SD = 11.59) for CT. No significant differences were observed except forY-1 and Bioxcell extenders (P < 0.05). It can be concluded that both methods of lipid/glycerol homogenization can be successfully applied in the preparation of bull semen extender. In addition, extensive lipid homogenization (10 cycles) produced more transparent extender that in turn improved visualization of sperm. Custom-made plant origin lipids homogenization may provide a valuable alternative for the preparation of extenders that more closely match the membrane composition of bull sperm cells and thus contribute to development of an efficient extender free of animal-origin components for bull semen freezing.

Effect of egg-yolk and its constituents on the respiration and fertilizing capacity of spermatozoa

1947 ◽  
Vol 37 (1) ◽  
pp. 69-76 ◽  
Author(s):  
J. Tosic ◽  
Arthur Walton
Keyword(s):  
Seminal Plasma ◽  
Phosphate Buffer ◽  
Respiratory Activity ◽  
Egg Yolk ◽  
Bovine Semen ◽  
Bull Semen ◽  
Main Factor

Respiratory activity of ejaculated bovine semen was studied in the presence of phosphate buffer (pH 7·4), egg-yolk medium or media containing different fractions isolated from the egg-yolk.Seminal plasma from centrifuged bull semen has very little respiratory activity as compared with whole semen. The main factor affecting the respiratory activity of the semen is the concentration of active spermatozoa, but the respiratory activity is not directly proportional to the concentration of spermatozoa, since it falls off with reduction of concentration.

scholarly journals Influence of Seasons and Extenders on Quality and Freezability of Gir Bull Semen under Middle Gujarat Climate

2018 ◽  
Vol 13 (03) ◽  
Author(s):  
D. V. Chaudhari ◽  
J. A. Patel ◽  
K. K. Hadiya ◽  
A. J. Dhami
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Winter Season ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Summer Season ◽  
Progressive Motility ◽  
Bull Semen

            The study was conducted to evaluate the seasonal influence (peak winter and summer) and the efficacy of three extenders (egg yolk based TFYG extender and egg yolk free soya bean based commercial extenders Optixcell and Andromed) on quality and freezability of Gir bull semen in Middle Gujarat. Semen ejaculates (6/bull/season, total36) revealed mean ejaculate volume 6.49±0.30 ml, sperm concentration1212.36±58.10 million/ml, progressive motility 74.17±0.78 %, live sperm 81.39±0.80 %, abnormal sperm 7.36±0.31 %, and sperm with intact plasma membrane 81.31±0.98 % and intact acrosome 94.81±0.24 %. Only the progressive sperm motility was significantly (P<0.05) higher(76.39±0.97 % vs. 71.94±1.00 %) with lesser sperm abnormality(6.17±0.37 % vs. 8.56±0.30 %) during winter than in summer. Semen samples split diluted with TFYG, Optixcell and Andromed extenders recorded the overall mean values of progressive sperm motility, livability, abnormality, plasma membrane integrity and acrosomal integrity during winter season as 77.87±0.51, 77.50±0.45, 5.56±0.20,76.02±0.81 and 94.35±0.29 on dilution; 72.41±0.51, 70.50±0.64, 5.96±0.26, 71.20±0.79 and 93.09±0.32 at pre-freeze stage; 41.30 ±0.94, 50.28±1.03, 9.15±0.31, 29.89±0.40 and 90.65±0.40 at post-thaw stage, respectively. The respective values in summer season were 72.13±0.60, 75.50±0.60, 7.48±0.25, 75.61 ±0.55 and 94.09±0.30 on dilution; 65.46±0.66, 69.41±1.05, 8.89±0.28, 69.70±0.66 and 92.63 ±0.33 at pre-freeze stage; 31.48±0.52, 45.09±0.85, 13.48±0.33, 26.85±0.71 and91.26±0.38 at post-thaw stage.  The overall mean sperm post-thaw motility/longevity at 0, 30, 60 and 120 min of incubation at 37°C was 41.20±1.51, 35.19±1.47, 28.80±1.75 and 17.50±1.47 % during winter season and 31.57±0.89, 26.20±0.77, 20.37±0.83 and 13.80±0.77% in summer season, respectively. The initial quality as well as freezability of semen in terms of motile, live, normal and HOS reactive sperm including post thaw longevity were better in winter season than in summer season. Further, the values of all the five semen quality parameters studied were comparatively better in Optixcell than TFYG and Andromed extenders with significant differences only in sperm progressive motility in both the seasons.The season x extender interaction was not significant for any of the sperm quality parameters studied.

76 THE EFFECT OF GLYCEROL CONCENTRATIONS IN EGG-YOLK CITRATE EXTENDER ON THE QUALITY OF CRYOPRESERVED NGUNI BULL SEMEN

2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
F. V. Ramukhithi ◽  
T. R. Netshirovha ◽  
C. Hlungwani ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Rural Areas ◽  
Egg Yolk ◽  
Positive Control ◽  
Negative Control ◽  
Glycerol Concentration ◽  
Bull Semen ◽  
Frozen Semen ◽  
Thawing Process ◽  
Motility Rate

There are bull shortages in South African poor rural areas. Artificial-insemination technology could play a significant role on breeding emerging farmer’s cattle. The objective of this study was to compare glycerol concentrations (0, 4, 8, or 12%) during freezing of Nguni bull semen to conduct AI in different villages. Semen was collected by electro ejaculator from 2 Nguni bulls of known and proven fertility. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories within 10 min after collections. Semen samples were pooled and evaluated by Sperm Class Analyser® and allocated randomly per treatment. Semen was then diluted (1 : 2 v:v) with egg-yolk citrate extender supplemented with either 0% (negative control), 4, 8, and 12% of glycerol concentration or AndroMed® (positive control). Semen samples were equilibrated for 4 h at 5°C. After equilibration period, samples were loaded into 0.5-mL straws and placed horizontally into the controlled rate (–5, –8, –10, –12, –15, –25, –35°C min–1) from 5°C until target temperature of –80°C is reached. The frozen semen straws were stored in a liquid nitrogen tank (–196°C) until thawing. Treatment means were separated using Fisher’s protected t-test least, and data are presented as mean ± SD. There was a significant differences (P < 0.05) between raw total sperm motility (83.3 ± 19.3) and frozen–thawed sperm with either 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), or 12% (61.5 ± 4.7) of glycerol and on AndroMed® (27.7 ± 17.8) group. Regardless of the glycerol concentrations used, the freezing-thawing process reduced (P < 0.05) the Nguni total sperm motility rate compared to uncryopreserved sperm (83.3 ± 19.3). In conclusion, egg-yolk citrate extender supplemented with 12% glycerol yielded a better (P < 0.05) total sperm motility rate (61.5 ± 4.7) as compared with the 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), and AndroMed® (27.7 ± 17.8) group. Further studies are required to test other levels of glycerol concentrations (>12%) on freezing Nguni semen and conducting AI.

21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen

2019 ◽  
Vol 31 (1) ◽  
pp. 136
Author(s):  
M. M. Tshabalala ◽  
K. A. Nephawe ◽  
M. L. Mphaphathi ◽  
C. M. Pilane ◽  
T. L. Nedambale
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Semen Parameters ◽  
Dna Integrity ◽  
Low Density ◽  
Bull Semen ◽  
Frozen Semen ◽  
Live Sperm

Egg yolk has been reported to have a beneficial effect on sperm quality following cryopreservation, and this led to its widespread use in semen extenders. However, egg yolk contains substances that inhibit respiration of sperm cells and diminish their motility rate. Moreover, it also contains low-density lipoproteins (LDL) that have a protective effect on sperm during the cryopreservation process. The objective of this study was improve cryopreservation of Nguni bull semen using egg yolk low-density protein. A total of 25 ejaculates were collected from 5 Nguni bulls aged 4 to 5 years using an electroejaculator during the natural breeding season. Collected raw semen samples were transported to the laboratory and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity before dilution. Semen was randomly diluted with a sodium citrate extender supplemented with 20% egg yolk (control) and with 6, 8, 10, and 12% LDL concentrations. The diluted semen sample groups were equilibrated for 4h at 5°C. Following equilibration, semen was loaded into 0.25-mL straws and frozen in a controlled-rate programmable freezer. The groups of semen straws were then plunged into LN and transferred into LN tanks (−196°C) for storage. The frozen semen straws per treatment group were thawed at 37°C and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity. Data were analysed with ANOVA using Stata V12 statistical software (StataCorp., College Station, TX), and treatment means were separated using Fisher’s protected t-test at the significant level of P&lt;0.05. Sperm motility and membrane integrity were significantly higher (P&lt;0.05) in frozen-thawed semen diluted with 8% LDL compared with the other concentrations. However, 6 and 8% LDL resulted in a significantly higher (P&lt;0.05) live sperm, DNA, and acrosome integrity. Frozen-thawed semen diluted with 10 and 12% LDL resulted in the lowest percentages of sperm motility, live sperm, plasma membrane, acrosome, and DNA integrity following cryopreservation. In conclusion, extender containing 8% LDL resulted in improved Nguni bull semen parameters such as sperm motility, viability, plasma membrane, acrosome, and DNA integrity following cryopreservation. Further studies are required to determine the fertilizing capacity of semen diluted and frozen with LDL in vitro and in vivo.

37 DOUBLE FREEZING AND THAWING OF NGUNI BULL SEMEN

2016 ◽  
Vol 28 (2) ◽  
pp. 148
Author(s):  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
T. R. Netshirovha ◽  
Z. C. Raphalalani ◽  
T. C. Chokoe ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Sperm Analysis ◽  
Treatment Groups ◽  
Bull Semen ◽  
Before And After ◽  
Repeated Freezing ◽  
Thawing Process ◽  
Marked Decline

Indigenous bulls semen are important for conservation programs. The objectives of this study were to evaluate the effects of repeated freezing and thawing on sperm motility characteristics. Semen was collected from 4 Nguni bulls by means of electro ejaculator and stored in a thermo flask (37°C). Sperm total motility, progressive and nonprogressive motility, and velocity were assessed using computer-aided sperm analysis before and after freezing. Semen was then diluted with egg yolk citrate extender (fraction A), then followed by 12% of glycerol + egg yolk citrate extender (fraction B, Seshoka et al. 2012). Diluted semen samples were equilibrated for 4 h at 5°C. After the equilibration period, samples were loaded into 0.25-mL straws and transferred into a controlled rate programmable freezer. After the target temperature of –130°C was reached, semen straws were stored in a LN tank (–196°C). After 3 months of storage, straws were thawed at 15°C (first and second freezing and thawing followed the same process) for 5 min and further evaluated post-thawed at 0 and 15 min during incubation at 15°C. Treatment means were separated using Fisher’s protected t-test least. No significant differences were recorded between the raw semen total sperm motility percentage (93.2%) and first frozen-thawed at 0 min (82.6%), with the total sperm motility rate recovery of 88.6%. In addition, there was a marked decline recorded in sperm total motility during the first frozen-thawed at 15 min (77.6%), second frozen-thawed at 0 min (31.3%), and second frozen-thawed at 15 min (30.1%; P < 0.05). The sperm curvilinear velocity and average path velocity was reduced following first frozen-thawed (P < 0.05) but remained constant and stable between the treatment groups (P > 0.05). In conclusion, the freezing-thawing process did not reduce the Nguni bull total sperm motility during the first freezing and thawing process, compared with raw semen. However, a drastic decline was recorded during the second freezing-thawing processes, compared with raw semen.

ASSESSMENT OF BOVINE SEMEN QUALITY BY RESAZURIN REDUCTION TEST

2016 ◽  
Vol 12 (1) ◽  
Author(s):  
Umesh Kumar ◽  
Prasanna Pati ◽  
A.P. Gawande
Keyword(s):  
Sperm Motility ◽  
Semen Quality ◽  
Sperm Concentration ◽  
Mean Value ◽  
Bovine Semen ◽  
Reduction Test ◽  
The Mean ◽  
Acrosomal Integrity

A study was conducted to assess the semen quality using Resazurin Reduction Test (RRT) in Jersey and Jersey cross bulls. A total 36 fresh ejaculates were collected for evaluation RRT, sperm motility, acrosomal integrity and sperm concentration. The mean value of RRT in bulls was 15.77±0.85 minutes.Statistical analysis revealed that RRT was highly significantly (p<0.01) and positively correlated with sperm concentration (r=0.769), sperm motility(r=0.858) and acrosomal integrity (r=0.515), hence it may be helpful in evaluating the quality of semen in bulls.

Expression study of fertility related genes with respect to sperm motility, HOST and acrosome integrity in bovine semen

2018 ◽  
Author(s):  
I. Borgohain ◽  
D. Dutta ◽  
P. Borah ◽  
D. Borah
Keyword(s):  
Sperm Motility ◽  
Adenylate Kinase ◽  
Expression Study ◽  
Osmotic Swelling ◽  
Bovine Semen ◽  
Bull Semen ◽  
T Complex ◽  
Complex Protein ◽  
Adenylate Kinase 1 ◽  
Fresh Semen

The present study was conducted for molecular evaluation of bull semen and to study its relationship with different semen characteristics. Fresh semen samples were collected from six breeding bulls. A total of six ejaculates were collected from each of six healthy breeding Jersey bulls at 4 days interval. Immediately after collection, the samples were subjected to physio-morphological and biomolecular evaluation. Percentage of Hypo-osmotic Swelling Test (HOST) positive spermatozoa and acrosome-intact sperms increased with an increase in initial sperm motility. Expression of Chaperonin Containing T-Complex protein 1, subunit 8 (CCT8) gene was found to be negatively correlated with sperm motility, whereas the expression of Adenylate Kinase 1 (AK1) gene did not show any significant relationship with sperm motility.

53 COMPARISON OF 4 DIFFERENT CRYOPROTECTANTS ON FREEZING NGUNI BULL SPERMATOZOA EVALUATED BY COMPUTER-AIDED SPERM ANALYSIS

2014 ◽  
Vol 26 (1) ◽  
pp. 140
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
T. L. Nedambale
Keyword(s):  
Sperm Motility ◽  
Artificial Insemination ◽  
Egg Yolk ◽  
Cleavage Rate ◽  
Sperm Analysis ◽  
Bull Semen ◽  
Significant Difference ◽  
Computer Aided ◽  
Freezing Curve ◽  
Motility Rate

Cryopreservation has been reported to damage approximately 40 to 50% of viable spermatozoa in bulls. It is critical to evaluate frozen-thawed spermatozoa with computer-aided sperm analysis (CASA) and find a suitable cryoprotectant for Nguni semen. The study was conducted to compare cryo-effectiveness of glycerol (GLY), dimethyl sulfoxide (DMSO), propanediol (PND), and ethylene glycol (EG) cryoprotectants at 12% concentrations during freezing of Nguni bull semen. Semen was collected from 18 stud Nguni bulls of proven fertility with the use of an electro-ejaculator. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories. Semen samples were pooled and sperm motility rate was evaluated using CASA. Semen was then diluted (1 : 2; vol : vol) with egg-yolk citrate extender supplemented with either 12% GLY, DMSO, PND, or EG. Semen samples were equilibrated for 4 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled rate programmable freezer using a customized freezing curve (from 5 to –5°C at 0.008°C min–1 and from –3 to – 130°C at 6°C min–1). Following thawing of semen, artificial insemination was conducted on 104 oestrus-synchronised Nguni cows. The IVF was also conducted on 120 oocytes to check the cleavage rate. Data were analysed using ANOVA. There was a significant difference (P < 0.05) between raw total sperm motility (94.70 ± 2.63) and frozen-thawed sperm total motility with GLY (77.80 ± 11.03), EG (20.35 ± 11.86), DMSO (15.68 ± 10.14), and PND (11.19 ± 11.27) groups. The pregnancy rate following artificial insemination was 75.9% and a total of 86.6% oocytes had cleaved after fertilization with frozen (12% GLY)/thawed semen. In conclusion, cryopreservation process reduced sperm motility and velocity rates, regardless of cryoprotectant. Egg-yolk citrate extender supplemented with 12% glycerol had recorded the highest post-thaw sperm motility rate.

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