scholarly journals Two Protocols of Cryopreservation of Goat Semen with the Use of Computer-Assisted Semen Analysis System

2007 ◽  
Vol 76 (4) ◽  
pp. 601-604 ◽  
Author(s):  
R. Kozdrowski ◽  
A. Dubiel ◽  
W. Bielas ◽  
M. Dzięcioł

The objective of the study was a comparison of two protocols of goat semen cryopreservation with the use of computer-assisted semen analysis system. Twenty ejaculates obtained with electroejaculation method were assessed. Each ejaculate was divided in half and frozen according to two protocols. In protocol I semen was centrifuged in order to remove its plasma and diluted in Tris buffer extender containing glucose, citric acid and glycerol with 20% addition of egg yolk. Protocol II did not include removal of plasma and the extender contained 1.5% egg yolk. It was shown that the removal of semen plasma improved motility of goat spermatozoa following freezing/thawing with respect to the following motility indicators: motility, average path velocity, amplitude of lateral head displacement at p < 0.05, and straight velocity, straightness and linearity at p < 0.01. In conclusion, the removal of semen plasma through centrifugation improved motility properties of goat semen following the freezing/thawing procedure.

2010 ◽  
Vol 22 (1) ◽  
pp. 204
Author(s):  
J. Dorado ◽  
M. J. Galvez ◽  
M. R. Murabito ◽  
S. Demyda ◽  
L. J. De Luca ◽  
...  

Tris-egg yolk-based diluents provide adequate cryoprotection for the sperm of most species. This study was conducted to compare the ability of Tris-glucose extender containing 2 different concentrations of egg yolk to maintain sperm motility and acrosome integrity of canine spermatozoa during 72 h of preservation. For this purpose, a total of 20 ejaculates from 4 clinically healthy dogs (2 Spanish Greyhound, 1 German Pointer, and 1 Crossbreed) were collected by digital manipulation. The sperm-rich fraction of each ejaculate was divided into 2 aliquots. Then, they were diluted in Tris-based extender and centrifuged at 700g for 8 min. Sperm pellets were resuspended in either Tris buffer added to 20% (EY20) or 10% centrifuged egg yolk (EY10) and cooled to 5°C over 72 h. The effects of these extenders on motility and acrosome integrity were assessed objectively using a computer-aided semen analyzer (Sperm Class Analyzer, Microptic SL, Spain) and Spermac® staining, respectively. Each cooled-rewarmed semen sample was evaluated after 24, 48, and 72 h of preservation. Sperm motion parameters shown by computer-assisted semen analysis (CASA) are progressively motile (PMS) and motile spermatozoa (MS), curvilinear velocity (CLV), average path velocity (APV), progressive speed (SLV), and lateral head displacement (LHD). Data were statistically analysed by ANOVA. Dependent variables expressed as percentages were arsine-transformed before analysis. Differences between mean values were evaluated by the Duncan method. Data were presented as mean ± SEM. Differences were considered significant when P < 0.05. Analyses were performed using the statistical package SPSS 12.0. A total of 98 172 motile sperm trajectories were analyzed by CASA: 52 259 in EY20 and 45 913 in EY10. After 24, 48, and 72 h of preservation, MS and PMS were statistically higher (P < 0.01) in EY20. No significant differences were found for LHD using either extender over a 72-h period. No significant differences were observed for CLV using either extender during the first 2 days. At Day 3, CLV data were significantly higher (P < 0.01) in EY20. Similarly, from Day 2, APV was significantly higher (P < 0.001) in EY20. After 24 h of preservation, SLV was statistically higher (P < 0.001) in EY10, whereas the opposite tendency was found at Day 3. No significant differences were observed for SLV using either extender after 48 h of preservation. During the first 2 days, acrosome integrity was statistically higher (P < 0.001) in EY20. At hour 72, higher acrosome integrity (P < 0.001) was observed in EY10. In conclusion, we have observed that the EY20 extender provided higher motility after 72 h of chilled preservation; however, the acrosome membrane integrity was better preserved in EY10.


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 ◽  
...  

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).


2015 ◽  
Vol 18 (4) ◽  
pp. 817-824 ◽  
Author(s):  
D. Gączarzewicz

Abstract The objective of the study was to evaluate the effect of different types of chambers used in computer-assisted semen analysis (CASA) on boar sperm concentration and motility parameters. CASA measurements were performed on 45 ejaculates by comparing three commonly used chambers: Leja chamber (LJ), Makler chamber (MK) and microscopic slide-coverslip (SL). Concentration results obtained with CASA were verified by manual counting on a Bürker hemocytometer (BH). No significant differences were found between the concentrations determined with BH vs. LJ and SL, whereas higher (p<0.01) values of this parameter were obtained with MK. Compared to MK and SL, significantly higher values were recorded in LJ for velocity (VCL and VAP) as well as amplitude of the lateral head displacement (ALH) and beat cross frequency (BCF), which was associated with significantly higher percentages of motile, progressively motile and rapidly progressive motile spermatozoa. Higher values for the linearity (LIN) and straightness (STR) of sperm movement were obtained for the analysis performed in MK and SL. In both these chambers, the results of all the linearity and kinetic parameters of sperm were similar (p>0.05). The results obtained show that CASA assessment of boar semen should account for the effect of counting chamber on the results of sperm motility and concentration, which confirms the need for further study on standardizing the automatic analysis of boar semen.


2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 115-115
Author(s):  
Carl R Dahlen ◽  
Sarah R Underdahl ◽  
Matthew S Crouse ◽  
Kacie L McCarthy ◽  
Cierrah J Kassetas ◽  
...  

Abstract Fifteen mature beef bulls (BW = 800.4 ± 17.4 kg) were used in a 112-d experiment to evaluate effects of divergent planes of nutrition on motility and kinematic properties of fresh and frozen-thawed semen. Bulls were ranked by BW and randomly assigned to one of two treatments: 1) managed on a positive plane of nutrition (POS, n = 8), or 2) managed on a negative plane of nutrition (NEG, n = 7). Bulls were fed a common diet, adjusted biweekly to achieve targeted weight loss or gain of 12.5% of original BW. On d 112, electroejaculation was used to collect 2 ejaculates from each bull, which were combined. An aliquot of fresh semen was evaluated via computer-assisted semen analysis (CASA; IVOS II, Hamilton Thorne, Beverly, MA, USA) for motility and kinematic properties. Remaining semen was extended and frozen. Frozen semen was thawed for 40 s and held in a heating block at 37°C, then evaluated via CASA at 0 and 3 h post-thaw. Data were analyzed in the MIXED procedure of SAS, with data for post-thaw analysis evaluated as repeated measures in time. Treatment did not influence ejaculate volume or concentration (P ≤ 0.19). In fresh ejaculates no impacts (P ≤ 0.29) of treatment were observed for motility or kinematic properties. In frozen-thawed ejaculates, however, bulls in the NEG treatment had greater (P ≤ 0.02) proportions of motile and progressively motile sperm compared with POS. In sperm classified as motile or progressively motile, NEG had greater (P ≤ 0.002) average path and straight line velocities, and greater (P ≤ 0.05) amplitude of lateral head displacement than POS. Treatment impacts observed in frozen, but not fresh, indicate that sperm metabolism, mitochondrial function, antioxidant capacity, or other factors may be influenced by plane of nutrition resulting in altered motility and kinematic properties.


2019 ◽  
Vol 50 (2) ◽  
pp. 96-103
Author(s):  
M. Ptáček ◽  
M. Stádníková ◽  
F. Savvulidi ◽  
L. Stádník

Abstract Kinematic parameters of thawed ram semen frozen under field conditions were analysed with the use of two commercial (egg yolk vs egg yolk free) semen extenders in different year-seasonal periods. The semen was collected from Suffolk (n = 2) and Charollais (n = 1) rams kept in private breeding farm on 3 test days (pre-mating, at mating, post mating) during year 2016. Two commercial semen extenders (egg yolk-based BullXcell® or egg yolk-free AndroMed®) were used for semen processing. Processed semen was frozen in 0.25 ml plastic cryostraws using the custom-made portable freezing box for ram semen cryopreservation under field conditions. Thawed semen characteristics were evaluated using computer-assisted semen analysis (CASA) system immediately after thawing and after 2 h of heat incubation (±38°C). Significantly higher total motility (+12.3%), straight line velocity (+5.6 μm s−1), and average-path velocity (+6.9 μm s−1) were detected for the semen processed and cryopreserved using egg yolk-based semen extender. Year-seasonal variation and introducing the ram to service had shown to have a significant effect on the cryopreserved ram spermatozoa. These preliminary results confirmed the feasibility of ram semen processing and cryopreservation under field conditions.


Zygote ◽  
2018 ◽  
Vol 26 (4) ◽  
pp. 301-307 ◽  
Author(s):  
José A. B. Bezerra ◽  
Andréia M. Silva ◽  
Patrícia C Sousa ◽  
Lívia B. Campos ◽  
Érica C. G. Praxedes ◽  
...  

SummaryThe aim of this study was to establish a functional freezing–thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen–thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.


2006 ◽  
Vol 18 (2) ◽  
pp. 151
Author(s):  
M. E. Carini ◽  
R. Cavia ◽  
G. Larraburu ◽  
G. M. Brogliatti

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.


2006 ◽  
Vol 18 (2) ◽  
pp. 150
Author(s):  
G. M. Brogliatti ◽  
G. Larraburu ◽  
R. Cavia ◽  
M. E. Carini

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.


2020 ◽  
pp. 2209-2218
Author(s):  
Fernando Evaristo da Silva ◽  
Jaqueline Candido Carvalho ◽  
Camila de Paula Freitas Dell'Aqua ◽  
Frederico Ozanam Papa ◽  
Marc Roger Jean Marie Henry ◽  
...  

The use of cooled semen in artificial insemination operations results in higher pregnancy rates than the use of frozen semen. This result seems to be related to the more severe damage triggered by the freezing process than that observed during refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a substance capable of preventing early sperm capacitation, a significant cause of the decreased pregnancy rate resulting from the use of frozen semen. The first objective of this study was to evaluate whether a commercial egg yolk diluent developed for frozen bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent in combination with sodium caseinate during the procedures of buffalo sperm cryopreservation using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membrane integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study, it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the harmful effects related to seminal cryopreservation.


2005 ◽  
Vol 17 (2) ◽  
pp. 156 ◽  
Author(s):  
G. Brogliatti ◽  
F. Garcia Migliaro ◽  
R. Cavia ◽  
G. Larraburu ◽  
A. Albrecht

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.


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