67 DELIVERING CHOLESTANOL OR DESMOSTEROL TO BULL SPERM MEMBRANES IMPROVES CRYOSURVIVAL

2008 ◽  
Vol 20 (1) ◽  
pp. 114
Author(s):  
E. A. M. Amorim ◽  
J. K. Graham ◽  
M. Meyers ◽  
B. Spizziri
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Plasma Membranes ◽  
Egg Yolk ◽  
Positive Control ◽  
Epifluorescence Microscopy ◽  
Osmotic Tolerance ◽  
Nitrogen Vapor ◽  
Bull Sperm ◽  
And Function

Altering the lipid composition of sperm plasma membranes affects sperm cryosurvival. Cryopreservation induces many stresses on the spermatozoa, including destabilization of the plasma membrane, which results in the loss of sperm motility and function. Treating bull spermatozoa with cholesterolloaded cyclodextrin (CLC) prior to cryopreservation increases sperm cryosurvival rates. This study compared the effect of adding other sterols, which should incorporate into the membrane and increase membrane fluidity at low temperatures, thereby increasing cryosurvival. Ejaculates from four bulls were divided into two experiments (E). In E1, ejaculates were extended with Tris, and then subdivided into four treatments: No additive (control), 1.5 mg CLC/120 million sperm (positive control), and 1.5 mg/120 million sperm in cyclodextrin pre-loaded with either cholestanol or desmosterol. Spermatozoa were incubated for 15 min at 22�C after which both the ability of fresh spermatozoa to bind to the zona pellucida (ZP) and chicken egg perivitelline membrane (EPM) and their osmotic tolerance were evaluated. In E2, sperm were diluted to 120 million cells mL–1 in a Tris diluent and treated as described for E1. Then, samples were diluted 1:1 (v:v) in Tris with 20% Egg Yolk (EY) and cooled to 5�C. After dilution 1:1 (v:v) with Tris containing 10% EY and 16% glycerol, samples were allowed to equilibrate for 15 min, and then were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min, and plunged into liquid nitrogen for storage. Straws were thawed and the motility and zona-binding ability were determined using a Hamilton Thorne Motility Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA) and epifluorescence microscopy, respectively. Treatment differences for sperm motility, osmotic tolerance, and zona binding were determined using analysis of variance. Treating spermatozoa with CLC resulted in more fresh bull spermatozoa binding to the EPM and ZP compared to cholestanolor desmosterol-loaded cyclodextrin-treated spermatozoa or control cells (P < 0.05). No differences were observed between EPM and ZP binding (P > 0.05). The percentages of total and progressively motile spermatozoa were higher for fresh samples treated with cholesterol-, cholestanol-, or desmosterol-loaded cyclodextrin than for control cells (P < 0.05) when spermatozoa were exposed to anismotic conditions, and then returned to isosmolality. After cryopreservation, the percentages of motile spermatozoa and number of spermatozoa binding to ZP were similar for spermatozoa treated with CLC (56% and 115 sperm/ZP) and cholestanol (53% and 108 sperm/ZP) compared to spermatozoa treated with desmosterol (42% and 86 sperm/ZP; P < 0.05). All treatments provided higher motility and binding efficiency than control spermatozoa (32% and 62 sperm/ZP; P < 0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival. Studies to determine if cholestanol affects sperm capacitation need to be conducted.

scholarly journals FREEZING CAPABILITY OF PASUNDAN BULL SPERM USING TRIS-EGG YOLK, TRIS-SOY, AND ANDROMED® DILUENTS

2017 ◽  
Vol 11 (1) ◽  
pp. 45-49
Author(s):  
Abdullah Baharum ◽  
R. Iis Arifiantini ◽  
Tuty Laswardi Yusuf
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Freezing Process ◽  
Sperm Abnormalities ◽  
Bull Sperm ◽  
Motility Of Sperm ◽  
Artificial Vagina

The aims of this study were to investigate the freezing capability of Pasundan bull spermatozoa in Tris-egg yolk (TEY), Tris-soy (TS), and AndroMed® as diluents. Semen were collected twice a week from four Pasundan bulls aged 3-5 years old using an artificial vagina and evaluated macro- and microscopically. Semen had ≥70% sperm motility, ≥800x106/mL sperm concentration, and less than 20% sperm abnormalities were divided into three parts and each of them diluted with TEY, TS, or AndroMed®. After an equilibration step at 5°C for four hours, diluted semen were packaged in 0.25 mL straw, frozen in liquid nitrogen for ten minutes and kept in liquid nitrogen container until examination. Motility test on fresh, diluted, equilibrated, and after-thawed semen was done using Androvision®. The results showed that after thawing motility of sperm diluted in AndroMed® (58.64±0.72%) was higher than in TEY (49.45±1.22%) and TS (39.34±6.33%). Sperm motility of Pasundan bulls diluted in these three diluents reduced around 33.27±2.45% during freezing process.

118 CRYOPRESERVATION OF CONVENTIONAL AND SEX-SORTED BULL SPERM USING A DIRECTIONAL FREEZING METHOD

2007 ◽  
Vol 19 (1) ◽  
pp. 176 ◽  
Author(s):  
H. Hayakawa ◽  
T. Yamazaki ◽  
M. Oshi ◽  
M. Hoshino ◽  
O. Dochi ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Interface Velocity ◽  
Egg Yolk ◽  
Sperm Viability ◽  
Freezing Method ◽  
Progressive Motility ◽  
Nitrogen Vapor ◽  
Bull Sperm ◽  
Directional Freezing ◽  
Acrosomal Integrity

The objective of this study was to find an optimal parameter (liquid-ice interface velocity; velocity) of the directional freezing method (Arav et al. 2002 Reprod. Nutr. Dev. 42, 583–586) for conventionally processed bull sperm stored in 0.5-mL straws. We also evaluated sex-sorted bull sperm frozen using this method. Experiment 1: Each single ejaculate from two Holstein bulls was split and processed in egg yolk citrate extender (2 steps, 6% glycerol final; EYC) or egg yolk Tris extender (1 step, 7% glycerol final; EYT1). After cooling and loading in 0.5-mL plastic straws, each batch of semen was frozen using a directional 0.5-mL straw freezer (MTG 550; IMT, Ltd., Ness-Ziona, Israel) in 6 different velocities (v2.0, v2.2, v2.4, v2.6, v2.8, v3.0; mm s-1). Ice seeding time was 45 s. Static freezing in liquid nitrogen vapor was used as the control. Sperm parameters were measured using computer-assisted semen analysis (CASA) at 0, 1, and 2 h after thawing. The thawed samples were also evaluated for sperm viability and acrosomal integrity by triple staining. The experiment was repeated 3 times. Data were analyzed by ANOVA. No significant differences in general and progressive motility were observed in each extender regardless of the freezing method. However, sperm viability and acrosomal integrity of sperm frozen in EYC were highest at v3.0 (73.2% and 84.7%, respectively) and were generally higher after MTG freezing (63.4 to 73.2% and 80.6 to 84.7%, respectively) than in the control (52.6% and 73.1%, respectively; P &lt; 0.05 or 0.01) except for v2.4 (54.5% and 70.8%). Sperm viability (74.6 to 77.9% vs. 63.2%) and acrosomal integrity (84.8 to 88.9% vs. 79.3%) in EYT after MTG freezing were also higher than in the control (P &lt; 0.01). Experiment 2: Two single ejaculates from a Holstein bull were flow cytometrically sex-sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) for X sperm. Each ejaculate was assigned to processing in EYC or egg yolk Tris extender (2 steps, 6% glycerol final; EYT2). Processed sperm was frozen using MTG 550 (velocity set at v3.0) or in liquid nitrogen vapor as control and analyzed as in Experiment 1. In the control, general (40.2% vs. 21.1%; P &lt; 0.01) and progressive (16.3% vs. 4.3%; P &lt; 0.05) motility in EYC were higher than in EYT2, respectively, at 0 h. In MTG freezing, motility (at 0 h, 42.0% vs. 28.3%; P &lt; 0.05), viability (60.5% vs. 40.8%; P &lt; 0.01), and acrosomal integrity (86.9% vs. 71.0%; P &lt; 0.05) in EYC were higher than in EYT2, respectively. But in experiment 2, there were no differences in motility, progressive motility, viability, and acrosomal integrity between MTG freezing and control. The above results suggest that MTG freezing improves membrane and acrosomal quality of bull sperm frozen in 0.5-mL straws. Faster interface velocity (3.0 mm s-1) seems optimal for the given condition. Egg yolk citrate extender seems to be beneficial for MTG freezing of sex-sorted bull sperm, but further studies using ejaculates from different bulls are required to confirm the apparent benefit.

80 TREATING BOAR SPERM WITH CHOLESTEROL IMPROVES CRYOSURVIVAL

2011 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
E. A. Moraes ◽  
C. A. A. Torres ◽  
J. K. Graham ◽  
P. L. Romualdo ◽  
P. S. Lopes
Keyword(s):  
Liquid Nitrogen ◽  
Zona Pellucida ◽  
Lipid Composition ◽  
Plasma Membranes ◽  
Egg Yolk ◽  
Glass Container ◽  
Epifluorescence Microscopy ◽  
Freezing And Thawing ◽  
Sperm Binding ◽  
Boar Sperm

Altering the lipid composition of plasma membranes not only affects the ability of sperm to capacitate and acrosome react, but also affects the way sperm respond to cryopreservation. When cyclodextrins are preloaded with cholesterol to form cholesterol-loaded-cyclodextrin (CLC) and then incubated with bull sperm before cryopreservation, higher percentages of motile and viable cells are recovered after freezing and thawing compared with control sperm. The amount of cholesterol in a membrane is important for maintaining its integrity during cryopreservation, and CLC alters the lipid composition of sperm, affecting their cryosurvival. This study evaluated the effect of adding cholesterol to boar sperm on cryosurvival rates and the ability of cryopreserved sperm to bind to the zona pellucida. Methyl-β-cyclodextrin was loaded with cholesterol as follows: 0.45 mL of cholesterol (200 mg mL–1 in chloroform) was added to 1 g of methyl-β-cyclodextrin dissolved in 2 mL of methanol, and the solution was stirred until clear. The mixture was poured into a glass dish and the solvents removed using a stream of nitrogen gas. The resulting crystals were allowed to dry for an additional 24 h, at which time they were removed from the dish and stored in a glass container at 22°C. A working solution of the cholesterol-loaded cyclodextrin was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C and mixing the solution briefly using a vortex mixer. Ejaculates from each of 8 boars were collected, diluted 1:1 in BTS® (Minitub, Brazil), and maintained for 2 h at room temperature. The ejaculates were then cooled to 15°C over 60 min. The ejaculates were then centrifuged at 400 × g for 10 min (at 15°C), the supernatant was discarded, and the sperm were suspended to 120 × 106 cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk). The sperm were divided into 2 treatments (T): T1 = control and T2 = 1.5 mg of CLC mL–1. The samples incubated for 15 min at 15°C, after which they were cooled to 5°C over 90 min and diluted 1:1 (v:v) with Freeze diluent (2.5 mL of lactose solution 11%, 6 mL of glycerol, and 1.5 mL of Orvus-es-Paste). The sperm were then packaged into 0.5-mL French straws and frozen in static liquid nitrogen vapor (4.5 cm above the liquid nitrogen) for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the efficiency of the sperm to bind to both the chicken egg perivitelline membrane (EPM) and porcine zona pellucida (PZP) were determined using epifluorescence microscopy. The post-thaw motility and binding efficiency of sperm to salt-stored EPM and PZP were analysed by analysis of variance. Boar sperm treated with CLC maintained higher post-thaw motility than control sperm (47 and 34%, respectively; P < 0.05) and had higher numbers of sperm binding to the PZP and EPM (101 sperm/EPM and 166 sperm/PZP) than control samples (77 sperm/EPM and 65 sperm/PZP; P < 0.05). In addition, sperm were easier to visualise on the EPM than the porcine zona pellucida. Adding CLC to boar sperm before cryopreservation increased the number of sperm surviving cryopreservation. Fapemig, CNPq, and CAPES from Brazil.

scholarly journals USE OF GLYCEROL AS CRYOPROTECTANTS IN FREEZING SENTUL CHICKEN SEMEN

2016 ◽  
Vol 1 (2) ◽  
pp. 6-13
Author(s):  
J. Junaedi ◽  
Raden Iis Arifiantini ◽  
Cece Sumantri ◽  
Asep Gunawan
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Recovery Rate ◽  
Cold Shock ◽  
Egg Yolk ◽  
Glycerol Concentration ◽  
Nitrogen Vapor ◽  
Frozen Semen ◽  
And Storage

On the freezing semen chicken cryoprotectants required to overcome the damage of spermatozoa due to cold shock. This study aims to get the best concentrations of cryoprotectants glycerol concentration of 5%, 7% and 9% in freezing Sentul chicken semen. The semen used in this study came from three chickens Sentul and be repeated nine times. Semen was collected by  messase methods for three times a week. Semen was evaluated macroscopic and microscopic. Furthermore spermatozoa diluted with egg yolk and the addition of three concentrations of cryoprotectants glycerol (5%, 7% and 9%). Semen diluted 0:25 ml is packed into straw. Then equilibrated at a temperature of 5°C for two hours. After equilibration to evaluate the motility and viability of spermatozoa. Furthermore, frozen in liquid nitrogen vapor for 10 minutes. Frozen semen is then stored in liquid nitrogen containers with temperature -196°C. After 24 hours, semen is thawed at 37°C for 30 seconds. The results showed that the percentage of sperm motility and viability of frozen semen cock Sentul using glycerol cryoprotectants 5% better P (<0.05) compared with the use of glycerol 7% and 9%. The use of glycerol 5% at this stage of equilibration and storage can reduce the damage of spermatozoa in the semen of chicken Sentul. Neither glycerol 5% could increase recovery rate after thawing

77 ADDITION OF CHOLESTEROL IN FRESH GOAT SPERM IMPROVES CRYOSURVIVAL RATES

2013 ◽  
Vol 25 (1) ◽  
pp. 186
Author(s):  
B. G. Silva ◽  
E. A. Moraes ◽  
C. S. Oliveira ◽  
W. D. Ferrari Junior ◽  
W. C. G. Matos ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Phase Contrast ◽  
Room Temperature ◽  
Petri Dish ◽  
Egg Yolk ◽  
Irreversible Damage ◽  
Hypoosmotic Swelling Test ◽  
Contrast Microscopy ◽  
Working Solution

Cryopreservation causes irreversible damage to goat sperm membranes, measured by a loss of motile and functional normal cells, compared with fresh sperm. The objective of this study was to determine if the addition of cholesterol-loaded cyclodextrin (CLC) to goat semen improved sperm cryosurvival. The CLC was prepared as described by Purdy and Graham (2004 Cryobiology 48, 36–45) with some modifications: 200 mg of cholesterol were dissolved in 1 mL of chloroform and 1 g of methyl-beta-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents allowed to evaporate on a warm plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C. Thirty ejaculates from 5 bucks were collected, diluted 1 : 1 in Tris diluent, divided into 7 equal aliquots, and centrifuged at 800g for 10 min. The sperm pellets were resuspended in Tris diluent, to which 0, 0.75, 1.5, 3.0, 4.5, 6.0, or 7.5 mg of CLC/120 million sperm were added. All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were then diluted with Tris-egg-yolk diluent containing 2% glycerol, and the sperm were packaged into 0.5-mL straws, frozen in static liquid-nitrogen vapour for 20 min, and plunged into liquid nitrogen. Straws were thawed in 37°C water for 30 s, extended in Tris, and analyzed using optic microscopy. To test thermal resistance, after thawing, 0.5 mL of semen from each treatment were placed in 1.5-mL Eppendorf tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm progressive motility. A hyposmotic test was also conducted by adding 10 µL of sperm to 2 mL of each solution and incubating them for 1 h/37°C. Sequentially, 20 µL of sperm was diluted in hypoosmotic solution (150 mOsm), and the samples were evaluated using phase-contrast microscopy. A total of 100 spermatozoa were counted in at least 5 different fields, and sperm tails were classified as either noncoiled or coiled. Data were analyzed using ANOVA, and treatment means were separated, using the SNK test at 5% probability. The sperm motility (50.4, 33.8, and 22.5%) was significantly higher for sperm treated with 0.75 mg of cholesterol after 0, 60, and 120 min of incubation after thawing, when compared with other treatments. No treatment differences in the hypoosmotic swelling test were observed. The addition of 0.75 mg of cholesterol to fresh goat semen improved sperm motility after cryopreservation for up to 3 h. Supported by FACEPE and CAPES.

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.

51 CHANGING ROOSTER SPERM MEMBRANES TO FACILITATE CRYOPRESERVATION

2012 ◽  
Vol 24 (1) ◽  
pp. 137 ◽  
Author(s):  
K. M. Tarvis ◽  
P. H. Purdy ◽  
J. K. Graham
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Membrane Composition ◽  
Sperm Analysis ◽  
Unsaturated Lipids ◽  
Multiple Comparison Test ◽  
Nitrogen Vapor

Cryopreservation damages rooster sperm membranes. Part of this damage is due to membrane transitioning from the fluid to the gel state as temperature is reduced. Some of this damage may be prevented by increasing membrane fluidity at low temperatures by incorporating cholesterol or unsaturated lipids into the membrane. Different concentrations of cholesterol-loaded cyclodextrins (CLC) and lipid-loaded cyclodextrins (LLC) containing 1,2-dilinoleoyl-sn-glycero-3-phosphocholine; 1,2-dilinoleoyl-rac-glycerol; and 1,2-dilinolenoyl-sn-glycero-3-phosphocholine were added to rooster sperm to determine if they improved cryopreservation. Osmotic stresses when cryoprotectants (CPA) are added to the cells before freezing and when the CPA are removed from cells after thawing also cause membrane damage. To minimize this damage, low molecular weight CPA with high membrane permeability were tested to determine their effectiveness for cryopreserving sperm. Rooster semen was collected from several birds, pooled and diluted to 800 million sperm mL–1 at 5°C in Lake's Low Temperature diluent (LLT). Sperm were treated with either LLC (0.25, 0.5, 1, 1.5, 2, 4 and 6 mg mL–1) or CLC (0.5, 1 and 2 mg mL–1) for 30 min. The sperm were diluted 1:1 with LLT containing 18% CPA, resulting in final CPA concentrations of 9%. The CPA tested were glycerol (G), methylacetamide (MA), dimethylformamide (DMF), methylformamide (MF) and ethylene glycol (EG). The sperm were frozen in liquid nitrogen vapor and stored in liquid nitrogen. Straws were thawed in 5°C water and sperm motility and membrane integrity analysed immediately. Sperm motility was measured using computer-assisted sperm analysis (CASA) and membrane integrity was analysed by flow cytometry using propidium iodide to detect cells with damaged membranes. Data were analysed by ANOVA and means separated using Student–Newman–Keuls multiple comparison test. Addition of LLC and CLC did improve sperm cryosurvival rates (P > 0.05). Using G as the CPA resulted in higher percentages of motile (54%) and viable (58%) sperm than MA (47 and 52%; P < 0.05), whereas DMF, EG and MF resulted in less than 45% motile cells (P < 0.05). In conclusion, altering sperm membrane composition using CLC and LLC did not improve post-thaw motility or viability in rooster sperm. Although MA did not protect the rooster sperm from cryodamage as effectively as G, future assays will need to determine the fertilizing capacity of sperm frozen using these CPA. We thank CSU-CVBMS for funding support.

scholarly journals The effect of freezing on cryosurvival of yak sperm

2018 ◽  
Vol 15 (2) ◽  
pp. 215-218
Author(s):  
S Deori
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Artificial Insemination ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Mean Values ◽  
Frozen Semen ◽  
Sperm Abnormality ◽  
Semen Characteristics ◽  
Live Sperm

A study was carried out to study the effect of freezing on cryosurvival of yak semen. Artificial insemination in yak is still in infancy. Semen cryopreservation and use of artificial insemination can be applied in yak husbandry for conservation and rapid multiplication of superior germplasm. Semen was collected from four adult yak bulls using artificial vagina method managed under uniform conditions. A total of 40 ejaculates comprising of 10 ejaculates each bull were collected following twice a week schedule and evaluated for fresh semen characteristics. The fresh yak semen characteristics viz. ejaculate volume (ml), mass activity (0-4), initial sperm motility (%), sperm concentration (x 106/ml), live sperm (%), sperm abnormality (%) and intact acrosome (%) were 3.10 ± 0.18, 3.53 ± 0.96, 83.89 ± 2.87, 1180.22 ± 42.32, 77.63 ± 4.23, 8.45 ± 3.33 and 93.61 ± 3.78 respectively. The ejaculates were diluted (1:10) with Tris extender consisting of 6.4 ml glycerol and 20 ml of fresh egg yolk. Straws were equilibrated at 5°C for 4 hours followed by exposure to liquid nitrogen vapour for 10 minutes and finally transferred to liquid nitrogen container for storage. The cryosurvival rate was studied after 7 days of storage in liquid nitrogen. The frozen semen was thawed in warm water (37°C) for 30 seconds for evaluation. Mean values of postthaw sperm motility (%), live sperm (%) and intact acrosome (%) in yaks were 55.67 ± 4.67, 65.62 ± 3.23 and 89.26 ± 3.67 respectively. In conclusion, yak semen has a better cryosurvival while freezing in tris extender with 6.4 per cent glycerol and 20 per cent egg yolk following an equilibration period of 4h.SAARC J. Agri., 15(2): 215-218 (2017)

scholarly journals CRYOPRESERVATION OF KAMPUNG ROOSTER SEMEN USING EGG YOLK DILUENT FROM FOUR TYPES OF POULTRY WITH DIFFERENT CONCENTRATIONS

2019 ◽  
Vol 13 (3) ◽  
Author(s):  
Khairuddin Khairuddin ◽  
Muhammad Erik Kurniawan ◽  
Soman Soman
Keyword(s):  
Liquid Nitrogen ◽  
Recovery Rate ◽  
Egg Yolk ◽  
Spermatozoa Motility ◽  
Chicken Egg ◽  
Nitrogen Vapor ◽  
Before And After ◽  
Egg Yolks ◽  
Chicken Egg Yolk

The aim of this study was to determine the type and the best concentration of egg yolk in maintaining the quality of kampung rooster spermatozoa during cryopreservation. This study used a completely randomized factorial pattern design with the first factor was the type of egg yolk (purebred chicken, kampung chicken, duck, and quail) and the second factor was the concentration of egg yolk (5%, 10%, and 15%). Semen was collected from twelve kampung roosters using massage method. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen with more than 70% motility was used in this study. The semen was diluted, packed in a ministraw, equilibrated, and frozen using liquid nitrogen vapor and stored in a liquid nitrogen container for 24 hours. Observation of spermatozoa motility was carried out in fresh semen, diluted semen, after equilibration and after thawing with four replications. The results showed that the type of egg yolk treatment had no effect (P0.05) on the recovery rate and motility of spermatozoa before and after cryopreservation, but egg yolk concentration had a highly significant effect (P0.01) on the quality of spermatozoa. Egg yolks in 10-15% concentration had spermatozoa motility and recovery rate higher than egg yolk with 5% concentration. In conclusion, purebred chicken egg yolk, kampung chicken egg yolk, duck egg yolk, and quail egg yolk each in diluent can be used to maintain the quality of kampung rooster spermatozoa at a concentration of 10-15% during cryopreservation.

Erratum to: 56 SINGLE LAYER CENTRIFUGATION THROUGH PURESPERM® 80 IMPROVES QUALITY OF CRYOPRESERVED DOG SPERMATOZOA

2013 ◽  
Vol 25 (1) ◽  
pp. 175
Author(s):  
L. Alcaráz ◽  
M. Hidalgo ◽  
M. J. Galvez ◽  
D. Acha ◽  
I. Ortiz ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Semen Quality ◽  
Semen Analysis ◽  
Gradient Centrifugation ◽  
Single Layer ◽  
Egg Yolk ◽  
Final Concentration ◽  
Viable Sperm

Density gradient centrifugation with PureSperm® (PureSperm® 40 + PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400 × 106 sperm mL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200 × 106 sperm mL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4 cm above the surface of liquid nitrogen vapors for 10 min, after which they were directly placed in liquid nitrogen. After 24 to 48 h of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P < 0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65 ± 0.05 v. 83.79 ± 0.13; percentage of progressive motile spermatozoa: 79.38 ± 6.66 v. 54.61 ± 16.11), morphology (86.45 ± 0.01 v. 83.51 ± 0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32 ± 0.04 v. 36.50 ± 0.17; percentage of viable sperm with an acrosome reaction: 2.81 ± 0.01 v. 9.74 ± 0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.

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