105 ALTERING SEMEN EXTENDER AND GLYCEROL CONCENTRATION TO OPTIMIZE RESULTS AFTER CRYOPRESERVATION OF EQUINE SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 170 ◽  
Author(s):  
M. Aceves ◽  
J. Scherzer ◽  
L. Ray ◽  
G. Heusner ◽  
R. A. Fayrer-Hosken
Keyword(s):  
Liquid Nitrogen ◽  
Survival Rates ◽  
Sperm Concentration ◽  
Skim Milk ◽  
Sample Volume ◽  
Glycerol Concentration ◽  
Motile Sperm ◽  
Slow Cooling ◽  
Progressive Motility ◽  
Semen Extender

After cryopreservation, spermatozoa from many stallions may have a lower capacity to fertilize an oocyte than fresh or cooled semen. The aim of this study was to evaluate a standard panel of semen extenders and varied concentrations of the cryoprotective agent (glycerol) to optimize sperm survival rates after cryopreservation. Semen was collected from Quarter Horse stallions (n = 3) from March to May 2006 (6 collections per stallion). Semen was filtered immediately after collection, and sample volume and sperm concentration were measured. A drop of raw semen was placed on two prewarmed slides to estimate the percentage of progressively motile sperm. The semen sample was then diluted to 100 � 106 spermatozoa/mL with a dried skim milk glucose extender (EZ Mixin Original Formula; ARS, Chino, CA, USA) or a chemically defined, milk-free diluent (INRA 96; IMV, Maple Grove, MN, USA). After 1 h of slow cooling and equilibration to 4�C, semen samples were centrifuged for 10 min at 400g. A defined volume of supernatant was removed, so that a concentration of 1000 � 106 spermatozoa/mL was obtained after resuspension of the sperm pellet. A 150-�L aliquot of semen was then added to specified quantities of the same semen extender used after semen collection and cryopreservation medium (Cryoguard�; Minitube, Verona, WI, USA) to obtain final glycerol concentrations of 2, 3, and 4%. This also gave a concentration of 100 � 106 spermatozoa/mL. After equilibration for 1 h at 4�C, spermatozoa were loaded into 0.5-mL straws and frozen in liquid nitrogen vapor. After 10 min, straws were plunged into liquid nitrogen. Semen was thawed at 37�C for 30 s and evaluated as prior to cryopreservation. Mean total semen volumes were 56, 11, and 60 mL in the 3 stallions. Their respective mean sperm concentrations were 124 � 106, 505 � 106, and 161 � 106 sperm/mL, respectively. Mean percentages of progressively motile sperm prior to cryopreservation were 64, 89, and 72%, respectively. With the paired Student's t-test, percentages of progressively motile sperm after cryopreservation were evaluated with respect to semen extender and concentration of glycerol used. Mean overall progressive motility of spermatozoa after cryopreservation differed significantly between the two extenders and was 46% for INRA 96 and 35% for EZ Mixin OF (P < 0.001). Using EZ Mixin OF as semen extender, the best mean post-thaw progressive motility was achieved with 4% glycerol (39%) and differed significantly from that with 2% glycerol (32%; P < 0.01). When INRA 96 was used (49% for 4%, and 42% for 2% glycerol), there was no difference. This results provide evidence that, during freezing of equine spermatozoa, there is a significant effect of the semen extender and the concentration of the cryoprotectant on post-thaw sperm motility. We therefore suggest mini-freezing trials prior to freezing large numbers of sperm to find the semen extender and glycerol concentration that provides optimal survival rates.

EVALUATION OF SEMEN FROM YOUNG BEEF BULLS

1968 ◽  
Vol 48 (3) ◽  
pp. 347-352
Author(s):  
H. B. Jeffery ◽  
R. T. Berg ◽  
R. M. Gratz
Keyword(s):  
Volume Concentration ◽  
Sperm Concentration ◽  
Progeny Test ◽  
Motile Sperm ◽  
Progressive Motility ◽  
Semen Volume ◽  
Beef Bulls ◽  
One Year

Semen was collected over a 30-day period from 18 yearling beef bulls, nine Herefords and nine hybrids. Average age at the start of the test was 380 ± 20 days. A total of 112 attempted collections resulted in 94 samples. Fifty percent of ejaculates were considered acceptable for freezing from Herefords and 79% from hybrids, respectively. The hybrid bulls produced significantly more semen per ejaculate (3.3 vs. 2.2 ml), higher sperm concentration (1092 × 106 vs. 657 × 106 per ml), higher sperm numbers per ejaculate (3,834 × 106 vs. 1,499 × 106) and higher total motile sperm per ejaculate (1,999 × 106 vs. 669 × 106). Bull-within-breed variance was significant for semen volume, concentration, initial motility and progressive motility. Progressive motility was in favor of semen from the hybrid bulls but was not statistically significant (48% vs. 36%). It is suggested that the superiority of the hybrid bulls was related to earlier onset of puberty. The trial also suggests that useable semen for progeny test purposes can be obtained soon after bulls reach one year of age.

scholarly journals Analysis of seasonal variability of sperm donors’ semen parameters

2018 ◽  
Vol 8 (3) ◽  
pp. 28-35
Author(s):  
Igor A. Korneyev ◽  
Ruslan D. Zasseev ◽  
Ol'ga B. Pashina ◽  
Ali E. Mamedov ◽  
Al'bert M. Dogov ◽  
...  
Keyword(s):  
Seasonal Variability ◽  
Reproductive Medicine ◽  
Sperm Concentration ◽  
Sperm Parameters ◽  
Semen Parameters ◽  
Motile Sperm ◽  
Sperm Number ◽  
Progressive Motility ◽  
Sperm Donors ◽  
The Mean

Introduction. The concept of seasonal variability of sperm parameters is controversial. However, it should be considered during medical evaluation and solicitation of sperm donors. Aim. To evaluate seasonal variability of sperm parameters from anonymous sperm donors in a reproductive medicine center. Materials and methods. A retrospective study of 1252 semen samples from 39 sperm donors (mean age 27.1 ± 3.9 years) in a reproductive medicine center in Saint Petersburg during the period from October 1, 2015 to October 1, 2017 was performed according to WHO 2010 recommendations. Semen volume, sperm concentration, total sperm number, progressive motility, and number of progressively motile sperm were analyzed. Results. Individual variability in semen parameters was high. The mean ejaculate volume in the summer months was higher than in the autumn, winter, and spring (t = 3.65, p < 0.001; t = 4.18, p < 0.0001; t = 1.92, p = 0.056 respectively). The lowest volume (2.83 ± 1.32 ml) was registered in January. The mean sperm concentration in summer was lower than in autumn, winter, and spring (t = 3.65, p < 0.001; t = 4.18, p < 0.0001; t = 1.92, p = 0.056 respectively). It was higher in winter than in spring and autumn (t = 2.54, p = 0.012; t = 1.72, p = 0.082 respectively). The highest mean sperm concentration was registered in January and the lowest in July (157.2 ± 46.6 and 131.9 ± 44.0 million sperm per ml, respectively). No significant seasonal differences were found in total sperm number, progressive motility, or number of progressively motile sperm (p > 0.1). Conclusions. The study suggests there is both individual and seasonal variability in sperm donors’ semen parameters. As several semen samples are needed to rate semen quality, we recommend that semen analysis for a potential sperm donor be performed in the summer months.

scholarly journals Application of high hydrostatic pressure (HHP) to improve cryopreservation of young bull semen

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Barbara Szczęśniak-Fabiańczyk ◽  
Piotr Gogol ◽  
Lechosław Gajda ◽  
Zdzisław Smorąg
Keyword(s):  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Motile Sperm ◽  
Cell Membrane Integrity ◽  
Progressive Motility ◽  
Bull Semen

Abstract The objective of the study was to determine the effect of high hydrostatic pressure (HHP) on quality of cryopreserved semen of young bulls. Semen for this study was collected from 8 bulls aged between 13 and 18 months at monthly intervals, from June to September. After collection, semen was diluted in a commercial Bioxcell® extender (one part at 1:1 and a second part to give a sperm concentration of 20 million/0.2 mL), filled into straws and treated with HHP at 30 MPa for 90 min. After HHP treatment, pre-diluted semen (1:1) was diluted to a sperm concentration 20 million/0.2 mL and filled into straws. In addition, part of the semen diluted to a concentration of 20 million/0.2 mL was not treated with HHP (control). All of it was held at +4°C and frozen in a freezer after 2.5-h equilibration. Semen was thawed in a water bath at 38°C and subjected to estimation of the percentage of motile sperm both subjectively and using a computer-assisted semen analyzer and cytometric assessment of sperm cell membrane integrity. Subjective motility and fast progressive motility were significantly higher with pre-diluted (1:1) and HHP treated semen compared to control (P<0.05). No significant differences were observed in percentage of membrane-intact spermatozoa between control and experimental groups. Additionally, the influence of HHP on the sperm of individual bulls was assessed. In bull number 2, the HHP treatment after semen pre-dilution significantly improved progressive motility from 54.1 to 63.4 percent (P <0.05). In bull number 4, the HHP treatment after semen pre-dilution significantly improved subjective motility, rapid motility and progressive motility by 12.5, 16.8 and 16.3 percent, respectively (P<0.05). No effect was seen for 6 bulls. It is concluded that for some bulls, the application of HHP before semen freezing may improve the cryopreservation outcome. However, this requires further research in this area, also to determine the fertilizing capacity of bull semen exposed to high hydrostatic pressure.

scholarly journals Effects of Sperm Parameters on Pregnancy Rate in Patients Undergoing Intrauterine Insemination

2020 ◽  
pp. 1
Author(s):  
Mehmet Solakhan ◽  
Mustafa Demir
Keyword(s):  
Intrauterine Insemination ◽  
Sperm Count ◽  
Sperm Concentration ◽  
Sperm Parameters ◽  
Motile Sperm ◽  
Progressive Motility ◽  
Age Related ◽  
Significant Difference ◽  
Sperm Volume ◽  
Infertile Couples

<p><strong>OBJECTIVE:</strong> In this study, the effects of sperm parameters on the success of intrauterine insemination were investigated. </p><p><strong>STUDY DESIGN:</strong> The data from 309 infertile couples who were admitted between 2012-2018 without a female factor were analyzed retrospectively and included in the study. After the administration of gonadotropin and hCG (5000-10000 IU), single insemination was performed in 36-40 hours in all cycles. All couples underwent routine infertility screening. The relationship between sperm parameters (motility, morphology, sperm count), patient age, duration of infertility with intrauterine insemination success was evaluated.</p><p><strong>RESULTS:</strong> There was no statistically significant difference between the two groups in terms of mean age and age related-parity. There was no statistically significant difference between male ages, liquefaction, and sperm volumes between the two groups (p=0.898, p=0.448, p=0.651). Before washing; There was a statistically significant difference between the sperm concentration, percentage of total motile sperm, percentage of progressive motility sperm, percentage of normal sperm morphology, and total sperm count between the two groups (p=0.0001, p=0.0001, p=0.0001, p=0.0001, p=0.0001). After sperm washing; the results were similar to those obtained before washing. While statistically significant difference was observed between sperm volume and sperm concentrations (p=0.023, p=0.018), no significant difference was observed between the two groups in total sperm count (p=0.612).</p><p><strong>CONCLUSION:</strong> As a result, during the application of intrauterine insemination to infertile couples, total motile sperm count, progressive motility sperm count ratio and high sperm ratio with normal morphology used in order to increase pregnancy success can be considered as criteria that increase the chances of success.</p>

113 Effect of antioxidants on motility and fertility of liquid-stored bovine sperm

2020 ◽  
Vol 32 (2) ◽  
pp. 183
Author(s):  
Y. Honkawa ◽  
T. Fujikawa ◽  
N. Miura ◽  
C. Kubota
Keyword(s):  
Embryo Development ◽  
Sperm Motility ◽  
Sperm Concentration ◽  
Frozen Storage ◽  
Egg Yolk ◽  
P Value ◽  
Motile Sperm ◽  
Progressive Motility ◽  
Bovine Sperm ◽  
Liquid Storage

It is difficult to maintain sperm in liquid storage for a long time, compared with permanent frozen storage in liquid nitrogen. Antioxidants have been reported to improve the quality and fertility of liquid-stored semen. In this study, we investigated whether antioxidants can extend the motility and fertility of frozen-thawed sperm in liquid storage. Frozen-thawed semen from one Japanese black bull (one ejaculate) was diluted in Tris-citrate-fructose (TCF) diluent with 10% (v/v) egg yolk to a sperm concentration of 1×107 spermmL−1. The antioxidants β-mercaptoethanol (βMe) and glutathione (GSH) were added independently, at various concentrations (0.1, 0.5, 1, and 5mM) to sperm suspensions, and these preparations were compared with Control (no added antioxidant). Sperm suspensions were packaged in centrifuge tubes and placed at 17°C in air and monitored daily until sperm motility had stopped (up to 14 days). Sperm motility was analysed by the Sperm Motility Analysis System (SMAS; Ditect Co. Ltd), and the percentage of progressively motile sperm (straight-line velocity (VSL) of &gt;25μm s−1; Grade A classified by WHO manual), compared with that recorded on Day 0 (100%), was determined each day. For evaluation of fertilizing ability, after incubation in liquid storage for 0, 3, 5, and 7 days, sperm were used for IVF with invitro-matured oocytes (30 oocytes per treatment, three replicates). Embryo development was recorded as the proportion of embryos that reached blastocyst by 8 days after IVF. Data for motility were analysed using one-way ANOVA with Tukey test, and embryo development using chi-squared test. A P-value&lt;0.05 was considered statistically significant. At 7 days, the percentage of progressively motile sperm was significantly higher for 0.5, 1, and 5mM βMe than for Control (30.8%, 48.1%, and 50.3%, vs. 0%, respectively). Treatments with 1 and 5mM βMe maintained some sperm progressive motility for 14 days (9.5% and 14.5%). Treatment with GSH showed the same trend at 7 days (32.2%, 36.3%, and 13.7% for 0.5, 1, and 5mM, vs. 0% for Control); 1 and 5mM GSH maintained sperm progressive motility over 10 days (24.8% and 4.4%). In both antioxidant treatments, embryo development was achieved with sperm stored for up to 5 days (Day 0 vs. Day 5 for 0.1mM βMe: 17.6% vs. 13.8%; for 1.0mM GSH: 26.0% vs. 6.7%; for Control: 17.6% vs. 0%). In this study, antioxidants extended both motility and fertility of frozen-thawed bovine sperm in liquid storage. This result suggests the possibility of application to AI using liquid-stored bovine semen.

PSIII-1 Influence of prenatal or postnatal exposure to gossypol from cottonseed meal on semen quality in commercial boars: a preliminary study on feral hog control

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 284-285
Author(s):  
Randi F Benefield ◽  
Richard A Mudarra ◽  
Tsung-Cheng Cheng Tsai ◽  
Christopher R Hansen ◽  
Charles V Maxwell ◽  
...  
Keyword(s):  
Cottonseed Meal ◽  
Semen Quality ◽  
Sperm Concentration ◽  
Sperm Cells ◽  
Motile Sperm ◽  
Postnatal Exposure ◽  
Treatment Groups ◽  
Progressive Motility ◽  
Feral Hog ◽  
Chi Square Analysis

Abstract The objective was to examine the influence of prenatal (Experiment 1) or postnatal (Experiment 2) exposure to gossypol from cottonseed meal (CSM) on semen quality in commercial boars. In Experiment 1, pregnant sows (n = 5) were fed a diet containing 0% (n = 1), 0.04% (n = 2), or 0.08% (n = 2) gossypol between d 56 and 86 of gestation. Boars (n = 11) born to sows in each treatment group (0% gossypol n = 3; 0.04% gossypol n = 4; 0.08% gossypol n = 4) were fed a common diet without CSM, and semen was collected at 269±2 d of age using a live sow in estrus. In Experiment 2, boars (n = 21) were fed a diet containing 0%, 0.02%, or 0.04% gossypol between 63±1 and 105±1 d of age (Initial BW: 19.85±0.43 kg). After the treatment period, boars were fed a common diet without CSM, and semen was collected at 238±7 d of age using a breeding dummy. Sperm cell concentration, percentage of motile sperm cells, and percentage of progressively motile sperm cells were analyzed using the MIXED procedure of SAS with treatment as a fixed effect in Experiment 1 and 2 and dam as a random effect in Experiment 1. In Experiment 2, semen was not successfully collected from every boar; therefore, chi-square analysis was used to assess semen collection status between treatment groups using the FREQ procedure of SAS. In Experiment 1, there was no difference in sperm concentration (P = 0.45), percent motility (P = 0.71), or percent progressive motility (P = 0.27) between treatment groups. In Experiment 2, there was no difference in sperm concentration (P = 0.72), percent motility (P = 0.17), or percent progressive motility (P = 0.87) between treatment groups. No difference was observed in boar collection status between treatment groups (P = 0.77). In conclusion, prenatal or postnatal exposure to gossypol from CSM did not influence semen quality in commercial boars.

scholarly journals 77 FREEZING OF MOUSE SPERM BY THREE DIFFERENT CRYOPROTECTANTS

2005 ◽  
Vol 17 (2) ◽  
pp. 188
Author(s):  
C.S. An ◽  
S.J. Uhm ◽  
D.H. Ko ◽  
H.M. Chung ◽  
S.-G. Cho ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Inbred Mice ◽  
Survival Rates ◽  
Skim Milk ◽  
Vital Staining ◽  
Mouse Strains ◽  
Storage Container ◽  
Vitro Fertilization ◽  
Mouse Sperm

The cryopreservation of sperm has contributed greatly to animal breeding and reproduction. This study was designed to examine the effect of raffinose, sucrose, and trehalose as cryoprotectants for freezing of mouse sperm. The cryoprotectant solution (CPA) consisting of 3% skim milk (Skim Milk dehydrated, Bacto, Difco, Seoul, Korea) as buffer or extender was prepared and supplemented with 0.3 M raffinose (D[+]raffinose pentahydrate, Sigma) or sucrose or trehalose as non-permeating cryoprotectants. Sperm samples for cryopreservation were collected from caudae epididymides and vas deferens of males of four mouse strains (ICR, FVB, C57BL/6, and CBA). Sperm samples from individual males were aliquoted in cryotubes, placed immediately in the vapor phase of a liquid nitrogen storage container for 10 min, and then plunged directly in liquid nitrogen. For thawing, frozen cryotubes were removed from liquid nitrogen and placed directly into a water bath kept at 37°C for approximately 2 min until the ice melted. Survival of mouse sperm was measured by vital staining. Survival rates of spermatozoa frozen and thawed in freezing solution supplemented with raffinose were higher (ICR: 51.0%, FVB: 27.6%, C57BL/6: 25.7%, and CBA: 23.3%) than those supplemented with sucrose (35.8, 19.6, 12.3, and 19.7%) or trehalose (16.0, 25.4, 25.3, and 24.7%), all respectively. Furthermore, in vitro fertilization of mouse oocytes with sperm frozen in raffinose gave cleavage rates of 61.1 (FVB: 217/355 eggs), 59.4 (C57BL/6: 165/278 eggs), and 57.1% (CBA: 144/252 eggs), respectively. These cleaved embryos (FVB: 163, C57BL/6: 137, and CBA: 134) were transferred to 7, 5, and 6 pseudopregnant females, respectively. A total of 4, 3, and 4 females in the respective strains became pregnant and delivered 17 (10.4%), 17 (12.4%), and 18 (13.4%) offspring, respectively. Our results suggest that raffinose is a good cryoprotectant for freezing of sperm for production of inbred mice.

scholarly journals Cryopreservation of Embryonic Axes and Axillary Buds of `Pineapple' Sweet Orange [Citrus sinensis (L.) Osb.] Encapsulated in Alginate Gel

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 440D-440
Author(s):  
Izulme R. Santos ◽  
Cecil Stushnoff
Keyword(s):  
Liquid Nitrogen ◽  
Cold Hardiness ◽  
Sweet Orange ◽  
Survival Rates ◽  
Germplasm Conservation ◽  
Axillary Buds ◽  
Embryonic Axes ◽  
Slow Cooling ◽  
Embryo Axes

Economically, citrus is the second most-important fruit crop grown worldwide; thus germplasm conservation of commercial cultivars, as well as of wild relatives, is essential. Presently, citrus germplasm has been conserved mainly in field genebanks. This approach is helpful; however, it is costly, exposes germplasm to climatic and biological hazards, and is not a long-term conservation system. Cryopreservation (conservation in liquid nitrogen, at –150°C to –196°C) is a technique that can ensure long-term storage of plant material. Attempts to cryopreserve citrus are restricted to a few reports, but the results obtained are encouraging. The basic purpose of this study is to define cryopreservation protocols for embryo axes and axillary buds of `Pineapple' sweet orange using the encapsulation-dehydration method. Embryo axes encapsualted in Na-alginate beads, precultured with high levels of sucrose and dehydrated over silica gel before freezing in liquid nitrogen had 60% survival. No survival was obtained for buds treated the same way, however buds isolated from plants acclimated at 0°C over a 30-day period survived exposure to –20°C when slow cooled at 2°C/hour. Additional experiments will combine cold acclimation, slow cooling and pre-treatment with sugars and other chemical compounds as an attempt to enhance cold hardiness of axillary buds and obtain survival after freezing in liquid nitrogen. Different approaches will be used to increase embryo axes survival rates.

scholarly journals Viability of carabao (Bubalus bubalis carabanensis) epididymal sperm from post mortem testes in semen extender at refrigerated temperature

2017 ◽  
Vol 4 (2) ◽  
pp. 5
Author(s):  
Lean Angelo B. San Diego ◽  
Marvin Bryan S. Salinas ◽  
Angelica C. Bumanlag ◽  
Marlon B. Ocampo ◽  
Lerma C. Ocampo
Keyword(s):  
Ambient Temperature ◽  
Sperm Concentration ◽  
Bubalus Bubalis ◽  
Post Mortem ◽  
Epididymal Sperm ◽  
Progressive Motility ◽  
Animal Genetic Resources ◽  
The Mean ◽  
Semen Extender ◽  
Excellent Source

Post mortem epididymal sperm (ES) is an excellent source of germplasm for conservation of animal genetic resources. In this study,  the viability of post mortem ES from carabao after collection at ambient temperature (AT) and after 24 hr and 48 hr of storage at refrigeration temperature (RT) were evaluated. ES were collected through slicing of epididymides from 7 carabaos. The mean ES volume was 0.4 ml with sperm concentration of 2.5×109 cells/ml. The mean percentage livability of fresh ES, after 24 hr and 48 hr of storage at RT were 81.93%, 65.93% and 43.7%, respectively. The mean percentage abnormalities of fresh ES, after 24 hr and 48 hr of storage at RT were 44.15%, 38.5% and 43.47%, respectively. The mean percentage motility of fresh ES, after 24 hr and 48 hr of storage at RT were 60.0%, 50.0% and 17.24%, respectively, after analysis through conventional means. Through CASA, the mean percentage motility was observed to be lower significantly at 26.12%, 37.94% and 18.32% for fresh ES, after 24 hr and 48 hr of storage at RT, respectively. The mean progressive motility of fresh ES, after 24 hr and 48 hr storage at RT were 10.02%, 16.47% and 10.87%, respectively. The results suggest that ES potential for use in fertilization studies remained viable when used immediately or after storage at RT for the first 24 hr.

scholarly journals Pengaruh Perbedaan Konsentrasi Gliserol dalam Susu Skim Kuning Telur untuk Proses Penyimpanan Sperma Beku terhadap Motilitas dan Viabilitas Spermatozoa Ikan Patin (Pangasius pangasius) [Effect Of Different Glycerol Concentration In Skim Milk And Egg Yolk Diluter For Storage Process Of Frozen Semen On Sperm Motility And Viability Of Catfish (Pangasius pangasius)]

2019 ◽  
Vol 6 (1) ◽  
pp. 1
Author(s):  
Bagus Rizki Novianto, Sudarno, Endang Dewi Masithah
Keyword(s):  
Sperm Motility ◽  
Sperm Concentration ◽  
Skim Milk ◽  
Egg Yolk ◽  
Fertilization Rate ◽  
Glycerol Concentration ◽  
Randomized Design ◽  
Frozen Semen ◽  
Egg Yolks ◽  
European Catfish

Abstract Needs of catfish seed always increase every years, especially for farming activities. Fulfillment demand of seed in large quntities and continuing are the main obstacle in the production process of catfish. This can be overcome by the process of cryopreservation. This process requires diluents and cryoprotectants to maintain the fertility of spermatozoa. Skim milk and egg yolks diluents has been used on fish because it was applied to carp and showing a good result of sperm motility and fertilization rate. Glycerol as cryoprotectant was reported effective to be used for European Catfish than the other kind cryoprotectant. This study aimed to determine the effect of different concentrations of glycerol in the diluent skim milk and egg yolk on the motility and viability of catfish (Pangasius pangasius) spermatozoa after freezing. This research method is the experiment with completely randomized design (CRD) as the experimental design. The treatment used is different glycerol concentrations, P1 (11%), P2 (13%), P3 (15%), and P4 (17%) and each of the treatment was repeated 5 times. The main parameters measured were motility (%) and viability (%) of spermatozoa. Supporting parameters include odor, color, pH, viscosity, sperm concentration and catfish (Pangasius pangasius). Analysis of the data using Analysis Of Variance (ANOVA) and to determine the best treatment performed Duncan's Multiple Range Test with 95% confidance interval. The results showed that the effect of the concentration of glycerol in the diluent skim milk and egg yolk was not significantly (P> 0.05) on the motility and viability of catfish (Pangasius pangasius) spermatozoa after freezing. Further testing needs to be done on the level of fertility of frozen sperm, as well as the growth rate of seeds produced from sperm frozen catfish (Pangasius pangasius) products

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