162 FREEZING OF SEMEN FROM ENDANGERED ASTURIANA DE LA MONTAÑA BULLS IN ZWITTERONIC LIPIDS-BASED EXTENDERS

2008 ◽  
Vol 20 (1) ◽  
pp. 161 ◽  
Author(s):  
C. Tamargo Miguel ◽  
S. S. Pérez-Garnelo ◽  
P. Beltrán Breña ◽  
A. T. Palasz ◽  
J. De la Fuente ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Semen Analysis ◽  
Egg Yolk ◽  
Semen Parameters ◽  
Computer Assisted ◽  
Progressive Motility ◽  
Bull Semen ◽  
Straight Line ◽  
Before And After ◽  
Arcsine Transformation

This experiment was designed to test the efficacy of 2 different preparation protocols of zwitteronic soyabean-origin lipids for the production of lipidsglycerol liposomes for use in bull semen cryopreservation. Lipids liposomes were prepared at 10% concentration in Tris buffer by 1. highpressure homogenization (Panda 2K, Parma, Italy) and then 8% glycerol were added, extender-1 (E-1); Lipids were homogenized together with glycerol, extender-2 (E-2). Bioxcell extender (E-3) was used as control. Semen was collected 3 times from 3 endangered Asturiana de la Monta�a bulls by the means of an artificial vagina. Ejaculates with at least 70% motility were processed further by a standard freezing protocol used in our AI station. Semen was diluted at 37�C with each of the 4 extenders to a concentration of 92 � 106 spermatozoa per mL, cooled to 4�C over 4 h, aspirated into 0.25-mL plastic straws (IMV Technologies, Aigle, France), frozen in a bio-freezer (IMV Technologies) in 3 steps from 4 to –140�C, and then plunged into liquid N2. Straws were thawed in a water bath at 37�C for 30 s. Sperm motility was analyzed microscopically immediately after collection, after dilution, and after 4, 24, 48, and 72 h of storage at 4�C. Post-thaw semen progressive motility was assessed microscopically, and sperm movement characteristics were analyzed by computer-assisted semen analysis (CASA) (SCA�, Microptic, Barcelona, Spain). Data were compared between extenders and bulls by 2-way ANOVA; percentages were transformed by arcsine transformation before ANOVA. Total and progressive sperm motility at 0 h after dilution ranged from 90 to 70% and was not different between extenders and bulls. There was no difference between bulls in total and progressive motility after 24 h of cold storage; however, both were significantly greater (P < 0.05) for Control (62.4 � 14.7 and 41.4 � 14.9) and E-1 (70.1 � 12 and 33.8 � 7.0) extenders than for the E-2 extender (22.5 � 17 and 1.2 � 1.3). Average post-thaw sperm motility was not different between bulls for either extender, but motility for Bioxcell (Control, 48.1 � 14.6%) and E-1 extenders (43.2 � 13.0%) were significantly greater (P < 0.05) than for E-2 extender (18.7 � 8.8%). There were no differences between bulls for all kinematic semen parameters: curvilinear (VCL), straight line (VSL), average path (VAP) velocities, linearity (LIN) and straightness (STR), evaluated by CASA before and after freezing; however, all were lower (P < 0.05) for the E-2 extender and not different between Control and E-1 extenders. Sperm movements derived from heads (VCL) and linearity of sperm(LIN), both closely related to field fertility, were in the range of 90.9 � 2.1 and 63.0 � 5.5 for E-3 (Control) extender; 99.1 � 3.4 and 49.4 � 3.5 for E-1; and 21.8 � 2.2 and 29.9 � 4.0 for E-2. In summary, zwitteronic soyabean lipid liposomes are an effective egg yolk substitute for the cryopreservation of Asturiana de la Monta�a bull semen; however, the homogenization protocol of the lipids-glycerol mixture must be improved.

55 EFFECT OF TREHALOSE FOR FROZEN–THAWED RAM SPERM MOTILITY PARAMETERS

2015 ◽  
Vol 27 (1) ◽  
pp. 120
Author(s):  
M. M. Toishibekov ◽  
M. T. Jazkbayev ◽  
B. B. Molzhigitov
Keyword(s):  
Sperm Motility ◽  
Semen Analysis ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Progressive Motility ◽  
Indigenous Sheep ◽  
Standard Tool ◽  
Freezing Curve ◽  
Ram Sperm

Computer-assisted sperm analysers have become the standard tool for evaluating sperm motility because they provide objective results for thousands of mammalian spermatozoa. Ram semen was collected using electro-ejaculation from 10 adult rams of Chingizskaya indigenous sheep breed. Motility was determined using computer-automated semen analysis (Hamilton Thorne Motility Analyzer, Beverly, MA, USA). Trehalose solution (0.375 M) was added to Tris-buffered saline solution to give the following trehalose extenders: 25, 50, 75, and 100% (vol:vol), and analysed for motility using computer-automated semen analysis. The sperm pellets were resuspended at 24°C in cooling extender – trehalose extenders of each concentration containing 5% egg yolk. The diluted semen was cooled to 5°C within 2 h. The semen was then further diluted 1 : 1 with freezing extender – each trehalose extender containing 1.5% glycerol to obtain a sperm concentration of 2.0 × 108 cells mL–1 – and then loaded into 0.5-mL straws. Straws were frozen using a programmable freezer with a freezing curve of 5°C to –5°C at 4°C per min, –5°C to –110°C at 25°C per min, and –110°C to –140°C at 35°C per min, and then the straws were plunged into liquid nitrogen for storage. Frozen samples were thawed in a 37°C water bath for 30 s and analysed for motility using computer-automated semen analysis. Statistical analyses were performed with a Student's test. The fresh semen samples showed the next results: motility 88.3 ± 2.4%, progressive motility 26.8 ± 6.9%, and progressive velocity 61.9 ± 4.2 μm s–1. Motility of the frozen-thawed spermatozoa was 63.6 ± 2.9% (25% trehalose), 55.6 ± 5.2% (50%), 32.4 ± 4.7% (75%), and 23.6 ± 3.2 (100%). Progressive motility was 15.6 ± 3.9% (25%), 13.7 ± 3.7% (50%), 4.5 ± 1.3% (75%), and 5.2 ± 1.3% (100%). Progressive velocity was 93.5 ± 8.3 μm s–1 (25%), 85.4 ± 8.1 μm s–1 (50%), 65.7 ± 6.1 μm s–1 (75%), 35.2 ± 3.3 μm s–1 (100%). Motility of the frozen-thawed spermatozoa significantly decreased with increasing concentrations of trehalose in the extender (P < 0.05). These preliminary studies showed that further research is needed of use trehalose for ram spermatozoa cryoconservation.

86 MOTILITY AND VIABILITY OF BULL SEMEN DURING 24 HOURS OF STORAGE AT 4°C

2006 ◽  
Vol 18 (2) ◽  
pp. 151
Author(s):  
M. E. Carini ◽  
R. Cavia ◽  
G. Larraburu ◽  
G. M. Brogliatti
Keyword(s):  
Storage Time ◽  
Semen Analysis ◽  
Computer Assisted ◽  
Bull Semen ◽  
Multiple Comparison Test ◽  
Straight Line ◽  
Semen Extender ◽  
One Way Anova ◽  
Velocity Average ◽  
Lateral Head

Currently, cryopreservation process of fresh bull semen is carried out between 3 and 6 hours of refrigeration at 4°C post-collection (Hafez, 1989). However, it is sometimes difficult when the cryopreservation process is not available at the site of collection. The objective of this study was to determine seminal motility and viability in samples maintained at 4°C during 24 hours. A total of 98 ejaculates from 23 adult bulls (Angus, Brangus, Braford and Hereford) were collected and diluted in a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) and stored at 4°C. Parameters of velocity average path (VAP, µm/s), velocity straight line (VSL, μm/s), amplitude lateral head (ALH, μm), linearity (LIN, %), percentage of rapid cells (RAPID, %), percentage of slow and static cells (SL/ST, %), and viability (VIA, %) were determined by Computer Assisted Semen Analysis (CASA, HTM-ceros 12.1, Berkeley, CA, USA). Measurements were done at 6, 9, 12, and 24 h. The obtained results were analyzed statistically with one-way ANOVA and Dunnet Multiple Comparison Test and are summarized in Table 1. There were no significant differences (P > 0.05) in the VAP, RAPID, or SL/ST during 24 h of storage at 4°C. Measurements were significantly different (P < 0.01) for VSL and VIA at 24 h. Measurements of ALH were increased from 12 h (P < 0.01) and consequently, LIN decreased at the same time (P < 0.01). These results suggest that there are no differences in velocity, except in VSL at the end of the storage time. The type of movement of the spermatozoa change, because ALH increases and the trajectory loses linearity. A decrease in viability suggests that from 24 h of storage, the membrane of the spermatozoa becomes more susceptible. More research needs to be done to evaluate the competence of this time-storage semen in the artificial insemination trial. Table 1. Parameters of motility and viability of semen maintained at 4°C during 24 h This research was supported by Centro Genético Bovino de EOLIA S.A.

84 EVALUATION OF BULL SEMEN AT 1 H VS. 48 H OF POST-FROZEN STABILIZATION TIME

2006 ◽  
Vol 18 (2) ◽  
pp. 150
Author(s):  
G. M. Brogliatti ◽  
G. Larraburu ◽  
R. Cavia ◽  
M. E. Carini
Keyword(s):  
Liquid Nitrogen ◽  
Artificial Insemination ◽  
Semen Analysis ◽  
Computer Assisted ◽  
Stabilization Time ◽  
Bull Semen ◽  
Straight Line ◽  
Semen Extender ◽  
Lateral Head

The process of cryopreservation of bull semen in liquid nitrogen at −196°C is usually carried out after 3 to 6 h of refrigeration at 4°C post-collection. To guarantee the quality of the final product, the frozen straws are evaluated after cryopreservation. The seminal samples are usually stabilized during 48 h before being analyzed (Hafez, Reproduction and Artificial Insemination in Animals, 1989); this would retard the possible commercialization. The objective of the present study was to determine motility parameters and viability of semen doses stabilized by 1 h or more than 48 h in liquid nitrogen at −196°C. A total of 122 ejaculated from 23 different adult bulls (Angus, Brangus, Braford, and Hereford) were evaluated in an artificial insemination center between January and April 2005. The semen was diluted in a semi-defined semen extender (Andromed, Minitub, Germany) and frozen in an automatic freezer (Digicool, IMV, France). Parameters of velocity average path (VAP, μm/s), velocity straight line (VSL, µm/s), amplitude lateral head (ALH, µm), linearity (LIN, %), percentage of rapid cells (RAPID, %), and viability (VIA, %) were determined by Computer Assisted Semen Analysis (CASA, HTM-ceros 12.1, Berkeley, CA, USA). The obtained results were analyzed statistically with T Student and are summarized in Table 1. The results indicate that there is no difference in the velocity of the spermatozoa evaluated 1 h or 48 h post-frozen. There is no difference in VAP, VSL, movement of amplitude lateral head (ALH), or linearity (LIN). The percentage of viable spermatozoa was not affected in either group. Statistical analysis indicates that there is no difference (P > 0.05) in any of the evaluated parameters. The results demonstrate that spermatic motility and viability of frozen bull semen could be evaluated before 48 h post-frozen. This allows reduction of the time between freezing and evaluation and immediate availability of the bull straws. Table 1. Parameters of motility and viability at 1 h vs. 48 h of post-frozen stabilization time This research was supported by Centro Genético Bovino EOLIA S.A.

137 Assessment of fertilizing ability of Merino ram semen cold stored up to 48h by heterologous IVF of bovine oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 194 ◽  
Author(s):  
D. A. Galarza ◽  
M. Ladrón de Guevara ◽  
P. Beltrán-Breña ◽  
M. J. Sánchez-Calabuig ◽  
A. López-Sebastián ◽  
...  
Keyword(s):  
Semen Analysis ◽  
Skim Milk ◽  
Semen Parameters ◽  
Computer Assisted ◽  
Cleavage Rate ◽  
Straight Line ◽  
Fertilizing Ability ◽  
Pronuclear Formation ◽  
Bovine Oocytes

The use of cold-stored ram semen has been applied in sheep AI programs, because it preserves its fertilizing ability similar to fresh. Besides, the heterologous IVF has been successfully employed to assess semen fertilizing ability in several species. Hence, we aimed to evaluate the fertilizing ability of ram semen cold stored up to 48h at 5°C by assessing heterologous IVF using bovine oocytes. Fifteen pools of 3 normospermic Merino ram (2-7 years) ejaculates were collected using artificial vagina, diluted to 200×106 spermatozoa mL−1 with ultra-heat-treatment-based extender (skim milk-6% egg yolk) and cold stored up to 48h. In vitro matured zona-intact bovine oocytes were subjected to heterologous IVF using fresh semen (FS, n=707), semen cold stored to 24h (CS24, n=832) or semen cold stored to 48h (CS48, n=611). In parallel, homologous IVF (control, n=1356) and parthenogenesis (parth control non-fertilized oocytes, n=334) were performed. Ram non-selected and selected (BoviPure, Nidacon International, Mölndal, Sweden) semen parameters were evaluated by computer-assisted semen analysis. Sperm-oocyte interaction was assessed at 2.5h post-insemination (hpi) by evaluating the number of bound spermatozoa, whereas penetration and polyspermy were evaluated after 12 hpi. Presumptive zygotes were fixed and stained with Hoechst 33342 at 18, 20, 22, 24 and 26 hpi to assess pronuclear formation using phase contrast and confocal microscopy. Cleavage rate was evaluated in all groups at 48 hpi. Data obtained from 5 replicates were analysed using one-way ANOVA. Data was expressed as mean±standard error of the mean. In terms of sperm storage time, non-selected semen showed a significant decrease (P&lt;0.05) for CS24 and CS48 compared with FS on progressive motility [SPM (%): 52.30±4.1 and 36.9±5.5v. 71.3±1.6] and straight-line velocity (mm s−1: 132.2±6.1 and 109.7±6.3v. 176.7±4.3), respectively. However, selected semen showed a decrease (P&lt;0.05) only for CS48 when compared with CS24 or FS on SPM (35.6±3.9v. 56.1±6.91 and 59.3±2.6) and straight-line velocity (83.5±4.4v. 105.3±6.5 and 110±2.0), respectively. No differences were observed between heterologous IVF groups in all parameters evaluated. Homologous IVF showed a higher percentage of penetration only when compared with heterologous FS group (44.4±6.8v.12.5±4.5%; P&lt;0.01). The polyspermy was higher in heterologous CS24 group when compared with homologous IVF (11.4±3.4v. 3.8±2.2; P&lt;0.05). The homologous IVF group, as expected, showed the higher percentage of pronuclear formation at 18 hpi compared with heterologous IVF with FS (67.3±5.8v. 35.2±5.6%), CS24 (72.1±4.5 v. 37.2±5.7%) and CS48 (63.0±6.0 v. 27.0±5.6%), respectively (P&lt;0.001). Likewise, cleavage rate was higher in homologous group compared with heterologous IVF and parthenogenetic groups for FS (78.3±2.6.8v. 46.3±3.2 and 7.0±2.3%), CS24 (78.4±2.6 v. 48.3±3.2 and 4.9±2.0%), and CS48 (78.4±3.3 v. 43.3±3.5 and 4.3±1.2%), respectively (P&lt;0.001). In conclusion, Merino ram semen cold stored up to 48h maintains its fertilization ability to the same extend as fresh and can be used for sheep crossbreeding programs.

scholarly journals 12 CASA PARAMETERS OF FRESH BULL SEMEN COLLECTED BY ARTIFICIAL VAGINA OR ELECTROEJACULATION IN ARGENTINA

2005 ◽  
Vol 17 (2) ◽  
pp. 156 ◽  
Author(s):  
G. Brogliatti ◽  
F. Garcia Migliaro ◽  
R. Cavia ◽  
G. Larraburu ◽  
A. Albrecht
Keyword(s):  
Semen Analysis ◽  
Glass Tube ◽  
Computer Assisted ◽  
Bull Semen ◽  
Straight Line ◽  
Beef Bulls ◽  
Semen Extender ◽  
Artificial Vagina ◽  
Lateral Head ◽  
Motility Parameters

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Its greatest advantages are elimination of the subjective nature of routine semen evaluation and the addition of detailed motion analysis unquantifiable by visual examination. The objective of this study was to evaluate CASA motility parameters of fresh bull semen collected by artificial vagina (AV) or electroejaculation (EE) from a total of 56 beef different bulls. Semen samples from a total of 45 beef bulls were collected by AV from winter to the end of spring (740 collections), and from 11 beef bulls by EE (120 collections) in the same period. First and second AV collections were analyzed as individual data. EE collection was performed only one. Means and standard deviations for each characteristic were calculated, compared, and statistically analyzed. A sample of the collection was diluted 1:20 in a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) and held in a glass tube at 36°C for 5 min before analysis. The sample was loaded into 20-μm chambers, and six microscope fields from each chamber were analyzed. The following sperm motility parameters were determined with the Ceros 12.1 sperm analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) on at least 1000 spermatozoa:concentration (CONC), velocity average path (VAP), velocity straight line (VSL), curvilinear velocity (VCL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of rapid or statics cells. There were no significant differences (P > 0.05) in VAP, VSL, VCL, ALH, STR, LIN, and the percentage of rapid and static cells of semen collected by AV or EE. The concentration (sperm/mL) of the AV-collected sperm was significantly higher than for the sperm collected by EE. Results from the analysis indicate that semen collected by artificial vagina have motility characteristics similar to those collected by electroejaculation. More research needs to be done to evaluate motility parameters of frozen/thawed semen collected by electroejaculation and by artificial vagina. This research was supported by Centro Genético Bovino de EOLIA sa Argentina.

57 EFFECT OF CHOLESTANOL OR CHOLESTEROL ON THE MOTILITY OF FROZEN GOAT SPERMATOZOA AFTER A THERMAL RESISTANCE TEST

2014 ◽  
Vol 26 (1) ◽  
pp. 142
Author(s):  
B. G. Silva ◽  
E. A. Moraes ◽  
W. C. G. Matos ◽  
C. S. Oliveira ◽  
W. D. Ferrari Junior ◽  
...  
Keyword(s):  
Thermal Resistance ◽  
Liquid Nitrogen ◽  
Semen Analysis ◽  
Egg Yolk ◽  
Water Bath ◽  
Resistance Test ◽  
Computer Assisted ◽  
Progressive Motility ◽  
Working Solution ◽  
Analysis System

The objective of the present study was to determine the concentration of cholesterol or cholestanol-loaded-cyclodextrin that needs to be added to goat sperm before cryopreservation to optimize its survival. The cholesterol or cholestanol loaded methyl-β-cyclodextrin was prepared as described by Moraes et al. (2010 Anim. Reprod. Sci. 118, 148–154). A working solution of the cholesterol or cholestanol-loaded cyclodextrin was prepared by adding 50 mg of each one to 1 mL of TALP at 37°C and mixing the solution briefly using a vortex mixer. Ejaculates (n = 24) from 5 bucks were used for this experiment. Sperm from each ejaculate were diluted 1 : 1 (vol : vol) in Tris diluent (200 mM Tris, 65 mM citric acid, and 55 mM glucose) and centrifuged at 800 × g for 10 min. The pellets were resuspended to a concentration of 120 × 106 sperm mL–1 in Tris and subdivided into 7 aliquots of 5 mL each (600 × 106 total sperm). Sperm were treated in 7 treatment groups that received no additive (0 mg; control) or different levels of cholesterol or cholestanol (0.75, 1.5, or 3.0 mg/120 × 106 sperm). All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were diluted with Tris-egg yolk diluent containing 2% glycerol. The sperm were packaged into 0.5-cc straws and frozen in static liquid nitrogen vapor for 20 min and then straws were plunged into liquid nitrogen and stored until analysed for motility and thermal resistance test using a computer-assisted semen analysis system (CASA). Two straws from each treatment were thawed in a 37°C water bath for 30 s and extended in Tris. For the thermal resistance test, after thawing, 0.5 mL of semen from each treatment was placed in 1.5-mL tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm total and progressive motility using a computer-assisted sperm motion analyzer. A total of 200 spermatozoa were counted in at least 5 different fields. Data were analysed using ANOVA and treatment means were separated, using the SNK test at 5% probability. Cholesterol (0.75 mg; 46.7%) and cholestanol (1.5 mg; 40.5%) produced an increase in progressive motility compared with other treatments after 1 h of incubation (P < 0.05). However, cholestanol (0.75 mg; 39.5 and 31%) was higher for total and progressive motility after 3 h of sperm incubation compared with the control (27 and 17.8%; P < 0.05), respectively. The addition of 0.75 mg of cholestanol in fresh sperm before cryopreservation improved the motility of freeze-thawed goat sperm compared with cholesterol. Therefore, adding cholestanol to goat sperm membranes improved cell cryosurvival. Supported by Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

34 The effect of dilution method of beagle dog semen on the survival rate of cryopreserved spermatozoa after thawing

2019 ◽  
Vol 31 (1) ◽  
pp. 143
Author(s):  
S. W. Kim ◽  
C.-L. Kim ◽  
I. S. Jeon ◽  
Y. G. Ko ◽  
I.-S. Hwang
Keyword(s):  
Drug Testing ◽  
Semen Analysis ◽  
Egg Yolk ◽  
Control Group ◽  
Dilution Method ◽  
Computer Assisted ◽  
Motile Sperm ◽  
Optimal Method ◽  
Beagle Dog ◽  
Progressive Motility

The successful cryopreservation of spermatozoa of the beagle dog for AI is essential for the establishment of the genetic banks of drug detection dogs. The beagle dog is widely used for drug testing and chosen for breeding by breeders. However, the use of cryopreserved beagle semen is limited by the lower number of offspring of dog species. In this study, 3 highly trained beagle dogs were chosen and their semen was cryopreserved for the next generation. The effects of dilution methods of beagle semen were tested using a direct dilution method at RT and a 2-step dilution method at 5°C. As a control group, the effects of a direct dilution method of semen on the percentage of motile sperm and progressive motility were analysed by computer-assisted semen analysis system (SAIS, Korea), and abnormality of spermatozoa was examined by Diff Quik staining. A total of 9 samples from 3 dogs were extended in 4% glycerol containing Tris-egg yolk diluents at approximately 22 to 25°C. The diluted semen was cooled to 5°C within 2h. The packed 0.5-mL straws were placed 5cm above the surface of LN for 10min and then plunged in. A 2-step dilution method was conducted using the same procedures of freezing, but the first dilution was done with glycerol-free diluent. After cooling to 5°C within 2h, the second diluent with 8% glycerol was added to the same volume of diluted semen at 5°C and stabilised for 1h. After thawing for 45s at 37°C, the semen from the 2-step dilution method showed the higher percentage of motile sperm (65.4±6% v. 45.3±8%; P&lt;0.05) and progressive motility (41.6±5.3% v. 32.3±3.7%; P&lt;0.05). However, the abnormalities between groups showed no differences. The results suggest that the optimal method for freezing beagle dog spermatozoa is a 2-step dilution process that consists of the first dilution at RT and the second dilution with glycerol at 5°C into diluted semen.

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.

scholarly journals 7 HETEROSPERMIC INSEMINATION AT TWO SPERM CONCENTRATIONS IN TIMED AI: CASA SEMEN PARAMETERS AND PREGNANCY RATES

2005 ◽  
Vol 17 (2) ◽  
pp. 154 ◽  
Author(s):  
A. Albrecht ◽  
R. Cavia ◽  
G. Larraburu ◽  
F. Garcia Migliaro ◽  
G. Brogliatti
Keyword(s):  
Artificial Insemination ◽  
Semen Analysis ◽  
Pregnancy Rates ◽  
Semen Parameters ◽  
Computer Assisted ◽  
High Concentration ◽  
Bull Semen ◽  
Motile Cells ◽  
Timed Artificial Insemination ◽  
Motility Parameters

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Heterospermic insemination has been used to increase pregnancy rates from low fertile bulls. The objective of this study was to evaluate, with the aid of CASA, heterospermic semen characteristics and pregnancy rates using different concentrations of bull semen in a timed artificial insemination protocol. Semen was collected from two bulls of known fertility by artificial vagina and all CASA motility parameters were evaluated individually and combined. Straws were filled using a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) as follows: single bull A and single bull B (12 × 106 of progressive motile cells after thawing); Mixed bull semen: A + B (12 × 106 of progressive motile cells after thawing) and Supermix bull semen: A + B (28 × 106 of progressive motile cells after thawing). All cows received a P4 intravaginal device (DIB, Syntex, Argentina) and 2 mg of EB i.m. (Syntex) on Day 0, 500 mg cloprostenol (Estroplan, Syntex) at the time of DIB removal (Day 8), and 1 mg EB i.m. on Day 9. Fixed-time insemination (FTAI) was performed at 52 to 56 h after DIB removal. A total of 249 cows were randomly allocated to be inseminated with bulls A and B (n = 76), with Mixed A + B (n = 87), and with Supermixed A+B at a high concentration (n = 86) by a single inseminator. Pregnancy rates were evaluated at 38 days after insemination by transrectal ultrasonography. Means and standard deviations or each characteristic were calculated, compared, and statistically analyzed. The following sperm motility parameters were determined with the Ceros 12.1 sperm analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) on at least 1000 spermatozoa: velocity average path (VAP), velocity straight line (VSL), curvilinear velocity (VCL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of rapid or static cells. There were no significant differences (P > 0.05) in VAP, VSL, VCL, ALH, STR, or LIN. There was a numerically higher percentage of rapid cells in the Supermix semen. Pregnancy rate from bulls A and B was 61% and from Mixed A + B 60%, while that from Supermixed A + B was 69%. Results from the analysis indicate that semen concentration is an important element to be considered in a timed artificial insemination program. Numerically higher pregnancy rates were obtained with double semen concentration in the straw. More research is required to evaluate the interaction between different breeds within a timed artificial insemination program. This research was supported by Centro Genético Bovino de EOLIA sa and Syntex sa Argentina.

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