scholarly journals Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection

2003 ◽  
Vol 15 (1) ◽  
pp. 19 ◽  
Author(s):  
Fiammetta Berlinguer ◽  
Sergio Ledda ◽  
Irma Rosati ◽  
Luisa Bogliolo ◽  
Giovanni Leoni ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Culture Media ◽  
Fluorescein Isothiocyanate ◽  
Spermatozoa Motility ◽  
Culture Systems ◽  
Fertilization Rates ◽  
Frozen Semen ◽  
Intracytoplasmatic Sperm Injection ◽  
Acrosome Integrity

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after IVF or intracytoplasmatic sperm injection (ICSI). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL−1 SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39°C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators’ observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For IVF, frozen–thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For ICSI, semen derived from the same culture systems as that for IVF was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the ICSI system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.

Liquid storage of ram semen in the absence or presence of some antioxidants

1996 ◽  
Vol 8 (6) ◽  
pp. 1013 ◽  
Author(s):  
WM Maxwell ◽  
T Stojanov
Keyword(s):  
Superoxide Dismutase ◽  
Glutathione Peroxidase ◽  
Intrauterine Insemination ◽  
Fertilization Rates ◽  
Liquid Storage ◽  
Semen Storage ◽  
Acrosome Integrity ◽  
Improvement In Survival ◽  
Fresh Semen

The antioxidants superoxide dismutase (SOD), catalase (CAT), cytochrome c (CHc) and glutathione peroxidase (GP) were added at various concentrations to Tris-glucose-yolk diluent (TGY), and their effects on motility, acrosome integrity and fertility of ram spermatozoa were assessed after extension and liquid storage. All the antioxidants improved the motility and acrosome integrity of spermatozoa, and a combination of SOD and CAT had an additive effect on the survival of spermatozoa stored at 5 degrees C but not at 25 degrees C. There was a linear improvement in survival of spermatozoa with increasing dose of antioxidants except for CAT for which doses higher than 200 U mL-1 were toxic. The proportion of oocytes fertilized in vitro declined with time of semen storage (P < 0.001), and was better for semen diluted with TGY containing SOD or CAT than TGY without antioxidants when stored for 7 days (116/246, 47% v. 25/79, 32%; P < 0.05) but not for 14 days (23/174, 13% v. 8/66, 12%). Fertilization rates were unaffected by the presence or absence in the diluent of CHc or GP. The proportions of ewes with fertilized ova and of recovered ova fertilized were better after insemination with semen diluted in TGY containing SOD and CAT than TGY without antioxidants when stored for 14 days (9/18, 50% and 20/40, 50% v. 2/13, 15% and 5/32, 16%; P < 0.05) but not for 7 days (9/20, 45% and 16/48, 33% v. 8/16, 50% and 24/41, 59%). Pregnancy rates were better after intrauterine insemination of ewes with fresh semen than stored semen (11/18, 61% v. 21/75, 28%; P < 0.01), and with semen stored in TGY containing SOD and CAT than in TGY without antioxidants (15/37, 41% v. 6/38, 16%; P < 0.05).

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection

1997 ◽  
Vol 9 (7) ◽  
pp. 665 ◽  
Author(s):  
M. C. Gómez ◽  
J. W. Catt ◽  
L. Gillan ◽  
G. Evans ◽  
W. M. C. Maxwell
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Acrosome Reaction ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Frozen Semen ◽  
Acrosome Integrity ◽  
And Control ◽  
Fresh Semen ◽  
Percoll Centrifugation

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitrofertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30°C or 39°C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30°C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
D. B. Koo ◽  
J. S. Park ◽  
K. K. Lee ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Developmental Rate ◽  
Treated Group ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Frozen Semen ◽  
Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

22 IN VITRO FERTILITY ESTIMATES USING SPERM KINETICS VARIABLES AND CONCEPTION RATES IN BEEF HEIFERS

2010 ◽  
Vol 22 (1) ◽  
pp. 169
Author(s):  
M. V. C. Ferraz Jr ◽  
R. S. Macedo ◽  
J. B. Barreto Filho ◽  
T. F. Silva ◽  
R. A. Braga Jr ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Ultrasound Images ◽  
Beef Heifers ◽  
Fast Evaluation ◽  
Spermatozoa Motility ◽  
Frozen Semen ◽  
Conception Rates ◽  
Inertial Moment

Sperm motility is a physical parameter evaluated in semen samples of the bull and is thought to be related to the fertility in the male. Despite being a characteristic of simple and fast evaluation, motility estimates involve subjective components when analyzed by light microscopy (LM) that might restrain their evaluation in some conditions. Moreover, in some species, poor correlations were observed between spermatozoa motility and semen fertility. The incidence of coherent light in a semen sample generates a phenomenon called biospeckle (BSL) that is capable of measuring the kinetics activity of the spermatozoa. In this work the relationship among sperm kinetics variables evaluated by LM and BSL and conception rates in beef heifers was investigated, with the purpose to predict frozen semen fertility when laser light is used. Sixty semen samples of 6 mature AI donor bulls (Bos taurus indicus) herein named A, B, C, D, E, and F were thawed at 37°C for 30 s in a water bath and evaluated by LM and BSL. In LM evaluation, an index (IND) was proposed to group together, in a single mean estimate, the spermatozoa motility (M: % scale) and velocity (V: 1 to 5 scale) according to the equation IND = [V × 20 + M]/2. In the BSL evaluation, each sample was illuminated (n = 10 per bull) by a laser beam (He-Ne, 632 nm and 10 MW) for 40 s and a mean inertial moment (IM) was obtained for each donor bull. These semen samples were used in an AI program of beef heifers (n = 166) clinically examined for reproductive health, body condition, and weight. Pregnancy diagnosis was done by ultrasound images (Falco 100, 6 MHz, Pie Medical, Crawley, UK) 28 days after insemination. Fertility estimates were done by the generalized linear model using logistic regression (stepwise methodology), generating an equation to predict the conception rate of the semen, the variables of which were IM and IND. Results of the predicted conception rates (pCR) using IM and IND and the observed conception rates (oCR) were A (0.5490; 0.57), B (0.6483; 0.68), C (0.7108; 0.71), D (0.4552; 0.28), E (0.4797; 0.54), F (0.3825; 0.47), respectively. Positive correlations (P < 0.05) were observed between pCR and oCR (r = 0.79) showing a similar behavior between in vitro and in vivo estimates. Results of this work showed that there is a high correlation between spermatozoa kinetics and semen fertility in the bull and that BSL motility analysis could be used as an approach to evaluate the fertility of semen samples. Financial support: FAPEMIG grant EDT 94/07, CNPq.

scholarly journals In vitro addition of docosahexaenoic acid improves the quality of cooled but not frozen–thawed stallion semen

2017 ◽  
Vol 29 (10) ◽  
pp. 2021
Author(s):  
D. M. Silva ◽  
S. A. Holden ◽  
A. Lyons ◽  
J. C. Souza ◽  
S. Fair
Keyword(s):  
Lipid Peroxidation ◽  
Vitamin E ◽  
Docosahexaenoic Acid ◽  
Membrane Fluidity ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Acrosome Integrity

The aim of the present study was to assess the effect of the addition of docosahexaenoic acid (DHA) on the in vitro quality of cooled and frozen–thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (three ejaculates per stallion). Semen was diluted to 100 × 106 spermatozoa mL–1 with 0.02 mM vitamin E (VE) and 0, 1, 10 or 20 ng mL–1 DHA and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from three stallions was collected (three ejaculates per stallion) and frozen as in Experiment 1, but VE and DHA were added after thawing. TM and PM were assessed at 30, 60 and 120 min and viability, acrosome integrity and membrane fluidity were evaluated at 30 min. In Experiment 3, semen from five stallions was collected (one to three ejaculates per stallion), diluted to 20 × 106 spermatozoa mL–1 and stored at 4°C. After 1, 24, 48 and 72 h, TM, PM, viability, membrane fluidity and lipid peroxidation were assessed. The addition of DHA had no effect on frozen semen (Experiments 1 and 2) but improved TM, PM and membrane fluidity in cooled stallion semen.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

2010 ◽  
Vol 58 (4) ◽  
pp. 465-474 ◽  
Author(s):  
Tamás Somfai ◽  
Yasushi Inaba ◽  
Yoshio Aikawa ◽  
Masaki Ohtake ◽  
Shuji Kobayashi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Culture Systems ◽  
Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

scholarly journals 316 SUPPLEMENTAL CYSTEINE PRESENCE DURING THE DECONDENSATION OF SPERM CHROMATIN IMPROVES FERTILIZATION AND BLASTOCYST FORMATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN PIGS

2005 ◽  
Vol 17 (2) ◽  
pp. 308
Author(s):  
M. Katayama ◽  
T. Cantley ◽  
A. Rieke ◽  
B. Day
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Morphological Changes ◽  
Formation Rate ◽  
Sperm Chromatin ◽  
Blastocyst Formation ◽  
Fertilization Rates ◽  
Duration Time

The effect of a cysteine supplement in culture media for oocytes matured in vitro after intracytoplasmic sperm injection (ICSI) on fertilization and embryo development were examined. In the first experiment, sperm injected oocytes were cultured in NCSU23 (control) or NCSU23 supplemented with 0.57–3.71 mM cysteine (0.57–3.71 Cys) for 12 h after ICSI, and then fixed to observe pronuclear formation. In the second experiment, to examine the appropriate duration time of cysteine supplement to support fertilization, sperm-injected oocytes were transferred into NCSU23 following culture in NCSU23 supplemented with 1.71 mM cysteine for 1, 2, 3, 4, 5, 6, or 9 h after ICSI, and then fixed at 12 h. At the same time, morphological changes of sperm heads in oocytes cultured in NCSU23 (1.71 Cys) were observed. In the third experiment, to examine the developmental ability of ICSI embryos fertilized in NCSU23 (1.71 Cys), sperm injected oocytes were cultured under the following conditions for a total of 168 h; NCSU23 (control), NCSU23 (1.71 Cys) for 3 h followed by transfer into NCSU23 (1.71 Cys-3 h), NCSU23 (1.71 Cys) for 12 h followed by transfer in NCSU23 (1.71 Cys-12 h), or NCSU23 (1.71 Cys) (1.71 Cys). Data were pooled from at least five replicates. Values in each replicate were analyzed using one-way ANOVA. Significance of differences was assessed by Student's t-test. Culture with several concentrations of cysteine for 12 h showed that 1.71–3.71 Cys significantly (P < 0.05) increased fertilization rates above controls or 0.57 Cys (56–60%, 35%, or 48%, respectively). Culture for several duration times with 1.71 Cys showed that fertilization rates increased as the duration time increased to 3 h which was significantly (P < 0.05) higher than controls (68% and 34%, respectively), and culture times of greater than 3 h did not increase fertilization rates (58–68%). At 3 h, 59% of oocytes cultured in NCSU23 (1.71 Cys) had decondensed sperm heads and 16% of those had enlarged sperm heads. At 6 h, 50% of oocytes cultured in NCSU23 (1.71 Cys) had male pronuclei. Blastocyst formation rate in 1.71 Cys-3 h was 29% which was higher than for controls (20%). On the other hand, 1.71 Cys-12 h cultures showed low blastocyst formation rates, and continuous culture in NCSU23 (1.71 Cys) for 168 h (1.71 Cys) significantly (P < 0.05) decreased blastocyst rates (16% and 7%, respectively). We found that the supplement of 1.71 mM cysteine to NCSU23 for culture of oocytes after ICSI improved fertilization rates. However, the presence of 1.71 mM cysteine for 12 h or longer after ICSI had adverse effects on embryo development. Since 1.71 mM cysteine supplement for 3 h after ICSI improved blastocyst formation with the same fertilization rates as when supplemented for 12 h, the presence of cysteine only during the decondensation of sperm chromatin was found to be associated with the improvement of fertilization and also the promotion of blastocyst formation.

scholarly journals Elisitör Olarak Kullanılan Çinko Sülfatın Biber Kalluslarının Süperoksit Dismutaz, Peroksidaz ve Toplam Fenolik Bileşikleri Üzerine Etkileri

2020 ◽  
Vol 8 (12) ◽  
pp. 2780-2784
Author(s):  
Cemil İşlek ◽  
Bengü Türkyılmaz Ünal ◽  
Sinan Aydın
Keyword(s):  
Cell Culture ◽  
Superoxide Dismutase ◽  
Enzyme Activities ◽  
Zinc Sulphate ◽  
Culture Media ◽  
Cell Suspension Cultures ◽  
Hot Pepper ◽  
Phenolic Substance ◽  
Peroxidase Enzyme

The amount of secondary metabolites can be increased with different elicitor applications in vitro. It has been determined that zinc sulphate significantly increases the amount of capsaicin in the cell culture of red hot pepper. It is important to determine how the metal applied as elicitor will have an effect on plant metabolism. In the study, it was aimed to determine the effects of zinc sulphate (ZnSO4) applied to the cell suspension cultures of pepper seeds at different concentrations (0.1 M, 0.2 M, 0.4 M) and for periods (24, 48, 72 hours) on the total protein and phenolic substance amounts, and superoxide dismutase-peroxidase enzyme activities of pepper calluses. It was observed that the amount of protein increased, superoxide dismutase and peroxidase enzyme activities decreased, and the total amount of phenolic substance increased especially in 72 hours of treatment where zinc was applied as elicitor. These results show that ZnSO4 can be used as an abiotic elicitor in plant cell culture media.

scholarly journals Mouse sperm delivery via cauda epididymis without transport medium

2016 ◽  
Author(s):  
Man-Xi Jiang ◽  
Mei-Shan Wang ◽  
Xiang-Hong Ou ◽  
Xue-Jin Chen ◽  
Yan Zhu
Keyword(s):  
Embryo Development ◽  
Sperm Motility ◽  
Room Temperature ◽  
Sperm Quality ◽  
Mouse Sperm ◽  
Spermatozoa Motility ◽  
Transport Medium ◽  
Fertilization Rates ◽  
Progressive Motility

This study was aimed to investigate the effects of room temperature (RT, 20-25 oC) and absence of medium during cauda epididymis transport on spermatozoa quality, fertility and embryo development. In the first experiment, fresh sperm from one side of cauda epididymis was used for in vitro fertilization, and another side was delivered at RT or 4-8 oC either with or without M2. In the second experiment, each side of cauda epididymis obtained from the same mouse was individually delivered at RT or 4 oC with or without M2. Finally, sperm motility, progressive motility scores and fertility of fresh spermatozoa or those from transported cauda epididymis, and IVF embryo development were evaluated. Progressive motility scores and fertilization rates were higher in fresh spermatozoa than transported sperm; sperm motility of transported cauda epididymis at 4-8 oC was comparable to fresh spermatozoa, but spermatozoa motility of transported cauda epididymis at RT was inferior to fresh spermatozoa. Spermatozoa motillty of transported cauda epididymis at 4-8 oC with transport medium was much higher than that without transport medium; absence of transport medium did not affect sperm motility of transported cauda epididymis at 4-8 oC but affected sperm motility of transported cauda epididymis at RT. Sperm quality from transported cauda epididymis can be efficiently kept at 4-8 oC, and cauda epididymis transport at 4-8 oC without M2 is more beneficial on keeping their fertility. Moreover cauda epididymis transport at RT without medium could sufficiently produce embryos for obtainning live offsprings.

scholarly journals The Effect of Centrifugation on Semen Quality of Peranakan Ettawah Goat's Post Thawing

2010 ◽  
Vol 4 (2) ◽  
Author(s):  
Indiah Indiah ◽  
Sri Wahjuningsih
Keyword(s):  
Semen Quality ◽  
Lower Layer ◽  
Vitro Fertilization ◽  
Spermatozoa Motility ◽  
Tukey Test ◽  
Abnormal Morphology ◽  
Frozen Semen ◽  
Experiment Method

The aim of the research was to determine the optimum of centrifugation on the quality of Peranakan Ettawah goat's spermatozoa prepared for in vitro fertilization preparation.The research of material was frozen semen ofPeranakan Ettawah goat produced by Artificial Insemination Center in Singosari with minimum post thawing motility in 40%. The method of the research was an experiment method with the four treatment, i.e: 1000 (P1), 1500 (P2), 2000 rpm (P3) of different centrifugation. The variable observes were spermatozoa motility, spermatozoa viability, and abnormal morphology of spermatozoa. Data obtain was analyzed statistically using Completely Randomize Design and continued with Tukey test. The result showed that on the upper layer of each treatment (P1, P2, and P3) obtained 55,72±3,55; 69,55±3,35; and 55,19±2,72% for spermatozoa motility, 62,99±5,87; 73,99±4,36; and 57,39±9,22% for spermatozoa viability, and 7,19±3,64; 8,84±2,65; and 6,40±3,00% for abnormal morphology of spermatozoa, descriptively. While on the lower layer the result showed 55,59±3,99; 68,63±3,88; and57,61±2,20% for spermatozoa motility, 70,05±7,47; 77,44±1,08; and 69,93±11,98% for spermatozoa viability, and 10,36±6,20; 9,55±3,27; and 8,09±2,80% for abnormal morphology of spermatozoa, desciptively. It was concluded that centrifugation in 1500 rpm showed the highest motility and viability on the upper layer and lower layer.

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