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 >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<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.

scholarly journals Effect of three commercial extenders on sperm motility and fertility in liquid ram semen stored at 15 °C or 5 °C

2019 ◽  
Vol 67 (3) ◽  
pp. 430-444
Author(s):  
Ander Arando ◽  
Juan Vicente Delgado ◽  
José Manuel León ◽  
Sergio Nogales ◽  
Francisco Javier Navas-González ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Soybean Lecithin ◽  
Egg Yolk ◽  
Kinematic Parameters ◽  
Progressive Motility ◽  
Head Displacement ◽  
Liquid Storage ◽  
Lateral Head

The effect of different extenders on sperm motility and fertility was evaluated during liquid storage of ram semen at 5 °C and 15 °C. The semen was collected, pooled and diluted in three commercial extenders: Inra 96® (INRA) based on skimmed milk, Biladyl® A fraction (BIL) based on egg yolk, and Ovixcell® (OVIX) based on soybean lecithin. Then, sperm motility was evaluated at 0, 6, 24, 48, 72 and 96 h. In order to evaluate fertility, samples stored at 15 °C were used after dilution in INRA and OVIX. Results showed that progressive motility was significantly higher up to 72 h of storage in sperm samples maintained at 5 °C in comparison with 15 °C, similarly for each tested diluent. When samples were stored at 5 °C in OVIX, kinematic parameters such as velocity (except curvilinear velocity, VCL), trajectory [linearity (LIN), straightness (STR), wobble (WOB)], amplitude of lateral head displacement (ALH) and beat/cross frequency (BCF) were higher than in INRA and BIL. No significant differences in pregnancy rate were detected between INRA (62.6%) and OVIX (58.9%). In conclusion, liquid storage at 5 °C with OVIX extender is an interesting option since non-animal components are used, and this extender offers similar in vitro and in vivo efficacy as other extenders containing animal components.

scholarly journals Effects of Media and Frame Rates on Hyperactivated Bovine Sperm Motility and an Analysis of Sperm Motility Subpopulation Structures in Sex-Sorted and Non-Sorted Semen

2021 ◽  
Author(s):  
Chihiro Kanno ◽  
Sun-Sik Kang ◽  
Kentaro Q. Sakamoto ◽  
Yojiro Yanagawa ◽  
Seiji Katagiri ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Discriminant Analysis ◽  
Sperm Motility ◽  
Computer Assisted ◽  
Motile Sperm ◽  
Progressive Motility ◽  
Bovine Sperm ◽  
Using Data ◽  
Effects Of Media ◽  
Oviductal Fluid

We attempted to establish an objective method to accurately evaluate the motility of bull sperm and examined the effects of media for sperm suspensions and frame rates on data of computer-assisted sperm motility analysis (CASA). Sperm incubated in Brackett and Oliphant medium (BO) more clearly showed hyperactivation-like motility than those in synthetic oviductal fluid. Sperm images captured at 150 frames per second (fps) showed a trajectory that was closer to the real pathway than those at other frame rates (30, 50, and 75 fps). We then examined the characteristics of sex-sorted and non-sorted semen using a cluster analysis followed by a discriminant analysis of sperm motility in BO at 150 fps. The results indicated that sex-sorted semen contained sperm with hyperactivation-like motility as the main subpopulation immediately after thawing and this subpopulation decreased after 2-h incubation. The main subpopulation in non-sorted semen had progressive motility that was maintained during incubation. In conclusion, usage of BO for sperm suspensions and capturing sperm motility at 150 fps by CASA were appropriate for evaluating bovine sperm motility. A discriminant analysis using data from a cluster analysis of motile sperm has the ability to accurately describe differences in the structures of sperm motility subpopulations.

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.

scholarly journals 10CASA EVALUATION OF SEXED AND NON-SEXED FROZEN BULL SEMEN

2004 ◽  
Vol 16 (2) ◽  
pp. 127 ◽  
Author(s):  
G. Brogliatti ◽  
G. Barreiro ◽  
G. Larraburu ◽  
A. Laborde
Keyword(s):  
Sperm Motility ◽  
High Speed ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Glass Tube ◽  
Egg Yolk ◽  
Motile Sperm ◽  
Bull Semen ◽  
Frozen Semen ◽  
Sexed Semen

Flow citometry cell sorting has been proven successfully to separate X and Y sperm; however, the technology is still too stressfull for the viability of the sorted semen. The objective of this study was to evaluate nonsexed and sexed frozen sperm motility characteristics using a CASA technology. Ejaculates from 4 different bulls (3 Holstein and 1 Angus) were collected, and processed as split non-sexed and sexed semen samples using Tris egg yolk extenders. X and Y sperm were separated using a high-speed sorter (SX Moflo). Cryopreservation was done at the same time under appropiate conditions using a programmed cryochamber. Thawing procedure was done at 37°C for 30s and a sample of each straw was placed in the evaluation chamber. The experiment was repeated twice and two chambers with 30 observations each were analyzed each time. Mean and standard deviation of each characteristic were calculated, compared and analyzed statistically. The sperm concentration was determined by means of a burker counting chamber. Sperm quality was determined at 0h after thawing, and later at 1h, 2h and 3h after incubation in a glass tube at 30°C. The following sperm motility parameters were determined with the Hamilton Thorne (HTM-ceros 12.1) on at least 1000 spermatozoa: velocity average path (VAP), velocity straight line (VSL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of progressively motile spermatozoa (PMS). Linearity of nonsexed spermatozoa was 53±3.5, 47±0.8, 43±7.8 and 42±4.5 for the 0h and the 3 test incubation times and 49.5±3.7, 51.2±3.7, 43.3±7.8 and 44.5±7.6, respectively, for sexed semen. There were no significant differences (P&gt;0.05) in the progressive velocity, track speed and linearity between sexed and nonsexed semen. The percentage of static cells was 33%, 30%, 47% and 50% for the 0h and the 3 test incubation periods; however, the percentage of static cells for the sexed semen was 53%, 71%, 77% and 82%, respectively. Results from the analysis indicate a significant increase (P&lt;0.01) in the number and the percentage of static cells with time. The lateral amplitude of sperm motility for nonsexed semen was 5.9±0.5, 6.8±0.8, 6.0±0.4 and 5.1±0.7, and for sexed semen 6.6±0.7, 6.8±0.4, 6.4±0.4 and 5.5±1.7, respectively. The percentage of progressively motile sperm was significantly different at 0 time 49.7±4.9 and 23.1±4.9 for nonsexed and sexed semen respectively. After 3 hours of incubation the percentage of progressively motile sperm was 38.7±10.2 and 3.7±3.2 for nonsexed and sexed semen, respectively. In conclusion, sexed frozen semen seems to have characteristics similar to those of normal nonsexed semen. However, a significant decrease in the percentage of progressively motile cells could affect pregnancy rates. More research needs to be done to detect differences between bulls and cryoprotectans.Research supported by Centro Genetico Bovino de EOLIA sa Argentina.

Dependence of the frequency of sperm defects and dimensions on sperm motility in ejaculates of Polish Landrace boars

2019 ◽  
Vol 15 (2) ◽  
pp. 33-45
Author(s):  
Maria IWANINA ◽  
Stanisław KONDRACKI
Keyword(s):  
Sperm Motility ◽  
Morphological Changes ◽  
Sperm Concentration ◽  
Sperm Cells ◽  
Motile Sperm ◽  
Morphometric Measurements ◽  
Progressive Motility ◽  
Significant Dependence ◽  
Normal Head

An attempt was made to determine the dependence of the frequency of sperm defects and dimensions on sperm motility in ejaculates of Polish Landrace boars. The study was conducted on 393 ejaculates collected from 33 Polish Landrace boars. Ejaculates were grouped according to the percentage of sperm with progressive motility, distinguishing ejaculates in which the percentage of motile sperm was 70% and 80%. In each ejaculate, the frequency of morphological changes in the sperm was determined and morphometric measurements of the sperm were made. Ejaculates with a higher proportion of sperm with progressive motility were found to contain more sperm. The ejaculate volume and sperm concentration in the ejaculate were not found to be directly associated with sperm motility. The frequency of primary defects was linked to sperm motility. Ejaculates with higher sperm motility contained fewer sperm with primary defects. The frequency of minor morphological changes, however, shows no significant dependence on sperm motility in the ejaculate. The primary morphological sperm defects most often found in ejaculates are a proximal droplet and the Dag defect. Both of these morphological forms are more common in ejaculates with lower sperm motility. The most common secondary sperm defects include sperm with a simple bent tail, sperm with a free normal head, and sperm with a distal droplet. These defects were not found to depend on sperm motility in the ejaculate. Sperm cells in ejaculates with greater sperm motility had slightly larger dimensions than sperm in ejaculates with lower sperm motility. Ejaculates with higher sperm motility are preferable for use in practice, not only because more insemination portions can be prepared from them, but also due to the lower frequency of primary defects.

scholarly journals Effect of fruit-juice on spermatozoa viability and lipid peroxidation of Red Sokoto bucks during liquid storage

2021 ◽  
Vol 41 (1) ◽  
pp. 16-27
Author(s):  
J. O. Daramola ◽  
T. A. Sorongbe ◽  
O. M. Onagbesan ◽  
A. V. Jegede ◽  
A. O. Ladokun ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Cell Damage ◽  
Egg Yolk ◽  
Protective Effects ◽  
Sperm Viability ◽  
Progressive Motility ◽  
Liquid Storage ◽  
Watermelon Juice ◽  
And Control

Antioxidants are linked with sperm viability because of their protective effects against cell damage during preservation. In order to enhance the life span of refrigerated buck semen, this study was carried out to determine the effect of fruit-rich antioxidants on spermatozoa viability and lipid peroxidation (LPO) of buck semen during liquid storage. Pooled semen from five Red Sokoto bucks was diluted with Tris-egg yolk based extender and supplemented each with juices from pawpaw tomato and watermelon at 0, 2.5, 5, 7.5 and 10/ 100 ml respectively. Following dilution, the semen samples were assessed subjectively after in vitro storage at 5°C for 24, 48, 72 and 96 hours as regards sperm motility, abnormalities, and acrosome status using a phase-contrast microscope. The concentration of malondialdehyde (MDA) as indices of lipid peroxidation (LPO) in the stored semen was measured in thiobarbituric acid reactive substances (TBARS) at 24, 48, 72 and 96 hours. The results showed highest progressive motility in watermelon juice at 2.5% (P<0.05) during the first 24 hours of storage while the lowest progressive motility was recorded at various levels of pawpaw juice (P<0.05). After 48 hours of storage, extender supplemented with watermelon and tomato juices had better progressive motility compared to control except 7.5% and 10%% of tomato juice (P<0.05). Irrespective of level of juice in the extender, the percentage of intact acrosome was similar among the various juices and control. The results showed that spermatozoa extended with watermelon juice had the lowest (P<0.05) percentage abnormality compared to other extenders at 24, 48, 72 and 96 hours of storage. Higher (P<0.05) percent spermatozoa abnormality compared to other fruit juices and control was observed at 72 and 96 hours of storage in spermatozoa extended with pawpaw juice. Significant reductions of MDA concentrations were achieved by addition of fruit-rich antioxidants to Tris-egg yolk based extender during the first 72 hours and the reduction was much pronounced in extender supplemented with pawpaw juice compared to control (P<0.05). The findings reveal that fruit-rich antioxidants from watermelon and tomato have protective ability to maintain sperm viability and to reduce concentration MDA of buck semen during liquid storage.

scholarly journals From Spermiogram to Bio-Functional Sperm Parameters: When and Why Request Them?

2020 ◽  
Vol 9 (2) ◽  
pp. 406 ◽  
Author(s):  
Rosita A. Condorelli ◽  
Aldo E. Calogero ◽  
Giorgio I. Russo ◽  
Sandro La Vignera
Keyword(s):  
Normal Form ◽  
Sperm Motility ◽  
Sperm Count ◽  
Semen Analysis ◽  
Sperm Concentration ◽  
Sperm Parameters ◽  
Sperm Parameter ◽  
Progressive Motility ◽  
Semen Volume ◽  
Chromatin Compactness

The aim of this experimental study was to evaluate whether infertile patients may benefit from the evaluation of bio-functional sperm parameters in addition to the conventional semen analysis. To accomplish this, we evaluated the correlation between conventional and bio-functional sperm parameters based on their percentile distribution in search of a potential threshold of these latter that associates with conventional sperm parameter abnormalities. The study was conducted on 577 unselected patients with infertility lasting at least 12 months. We identified cut-off values according to the median of the population for mitochondrial membrane potential (MMP), number of alive spermatozoa, and chromatin abnormality. High MMP (HMMP) (≥46.25%) was associated with sperm concentration, sperm count, progressive motility, and normal form. Low MMP (LMMP) (≥36.5%) was found to be associated with semen volume, sperm concentration, total sperm count, progressive motility, total motility, and normal form. The number of alive spermatozoa (≥71.7%) was associated with sperm concentration and progressive motility whereas abnormal chromatin compactness (≥21.10%) was associated with sperm concentration, total sperm count, and progressive motility. The data would suggest that, for every increase in the percentile category of sperm concentration, the risk of finding an HMMP≤46.25 is reduced by 0.4 and by 0.66 for a total sperm count. This risk is also reduced by 0.60 for every increase in the percentile category of sperm progressive motility and by 0.71 for total sperm motility. Each increment of percentile category of the following sperm parameter was followed by a decrease in the risk of finding an LMMP≤36.5: sperm concentration 1.66, total sperm count 1.28, sperm progressive motility 1.27, total sperm motility 1.76, and normal form 1.73. Lastly, the data showed that, for every increase in the percentile category of total sperm count, the risk of finding an abnormal chromatin compactness ≤21.10 is reduced by 1.25 (1.04–1.51, p < 0.05) and an increase of total sperm motility is associated with a reduced risk by 1.44 (1.12–1.85, p < 0.05). Results suggest a correlation between bio-functional and conventional sperm parameters that impact the sperm fertilizing potential. Therefore, the evaluation of bio-functional sperm parameters by flow cytometry may be useful to explain some cases of idiopathic male infertility.

284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)

2006 ◽  
Vol 18 (2) ◽  
pp. 249
Author(s):  
G. Magarey ◽  
J. Herrick ◽  
K. Thiangtum ◽  
W. Tunwattana ◽  
W. Swanson
Keyword(s):  
Sperm Motility ◽  
Culture Medium ◽  
Culture Media ◽  
Egg Yolk ◽  
Ovarian Follicles ◽  
Blastocyst Stage ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
International Transport

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P > 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P < 0.05) and declined faster (P < 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P < 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P > 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro. This work was supported by (NIH RR015388).

71 BOVINE SPERM DEATH KINETICS: CHANGES IN MOTILITY, ACROSOMES, AND PLASMA MEMBRANE

2013 ◽  
Vol 25 (1) ◽  
pp. 183
Author(s):  
M. Ahmad ◽  
N. Ahmad ◽  
A. Riaz ◽  
M. Anzar
Keyword(s):  
Plasma Membrane ◽  
Statistical Analysis ◽  
Sperm Motility ◽  
Propidium Iodide ◽  
Female Reproductive Tract ◽  
Semen Sample ◽  
Membrane Asymmetry ◽  
Progressive Motility ◽  
Bovine Sperm ◽  
Acrosomal Integrity

Extent and timing of alterations in structures and functions of sperm after its placement in the female reproductive tract are important for successful fertilization. To our knowledge, the few reports are available on the kinetics of alterations in bovine sperm structures and functions during pathway to their death. Therefore, the present study was conducted to determine the changes in motility, acrosome and plasma membrane asymmetry in fresh and frozen–thawed semen during incubation at 37°C over the period of 24 h. Semen was collected from 3 breeding beef bulls, pooled, and considered as one replicate (total replicates = 5). Each pooled semen sample was diluted in Tris-citric acid egg yolk glycerol extender (pH 6.8), cooled to +4°C over 90 min, and then cryopreserved by a programmable cell freezer. Fresh (pooled semen) and frozen–thawed semen were incubated at 37°C for 24 h. Each semen sample was evaluated for sperm motility with computer-assisted semen analysis and acrosomal integrity and plasma membrane asymmetry using fluorescein isothiocyanate-peanut agglutinin/propidium iodide and Annexin V/propidium iodide assays, respectively, at 0, 2, 4, 6, 12, and 24 h of incubation at 37°C, with a flow cytometer. Statistical analysis was conducted using PROC MIXED model in statistical analysis system as 2 (semen types) × 6 (times) factorial model, using time as repeated measure. Progressive motility was higher (P < 0.05) in fresh than in frozen–thawed semen until 6 h. Progressive motility declined (P < 0.05) below the threshold level (i.e. 30%) much later (12 h) in fresh as compared with frozen–thawed semen (2 h). However, acrosomal integrity and plasma membrane asymmetry deteriorated (P < 0.05) below threshold at the same time interval (2 h) in both fresh and frozen–thawed semen. Viable sperm (AN–/PI–) remained higher (P < 0.05) during the first 6 h in fresh than in frozen–thawed semen and declined (P < 0.05) below the threshold at 12 h in fresh and at 6 h in frozen–thawed semen. In fresh semen, the necrotic sperm (AN–/PI+) population increased (P < 0.05) over time and reached maximum (97%) at 24 h. In frozen–thawed semen, a mixed population of late apoptotic (53%) and necrotic (34%) sperm was found at 24 h. In conclusion, the alterations in sperm motility, acrosomes, plasma membrane integrity, and asymmetry were slower in fresh than in frozen–thawed semen. Fresh sperm followed necrosis and frozen–thawed sperm underwent necrosis and apoptosis-like pathways, respectively. This study was supported by the Canadian Commonwealth Scholarship Program by the Canadian Bureau for International Education (CBIE), and Agriculture and Agri-Food Canada.

scholarly journals Comparison of Extenders With the Addition of Egg Yolk for Cooling Alpaca Sperm Obtained From Deferent Ducts

2020 ◽  
Vol 7 ◽  
Author(s):  
Mariana Lucía Bertuzzi ◽  
Edita Yola Torres ◽  
Teodosio Huanca ◽  
Deborah Neild ◽  
María Ignacia Carretero
Keyword(s):  
Sperm Motility ◽  
Chromatin Condensation ◽  
Egg Yolk ◽  
Final Concentration ◽  
Sperm Parameters ◽  
Membrane Function ◽  
Progressive Motility ◽  
Evaluation Time ◽  
Viable Sperm ◽  
Acrosome Integrity

The use of non-commercial and commercial extenders for cooling alpaca sperm has already been reported, the latter showing certain advantages over the first. The Andromed® (AM) extender was created for use in ruminants and has also been tested in ejaculated and epididymal alpaca sperm. According to the manufacturer, this extender does not need the addition of egg yolk (EY); however, it is known that the addition of EY to some extenders improves the preservation of cooled sperm. The objective of this study therefore was to compare a non-commercial extender (Tris) with the addition of EY vs. the commercial extender AM with and without the addition of EY, for cooling alpaca sperm obtained from diverted deferent ducts. Fifteen pools of deferent duct sperm were formed using samples from two or three different males for each. Each sperm pool was evaluated and then divided into three aliquots that were diluted to a final concentration of 30 × 106 sperm ml-1 (0 h) with either: (1) Tris with 20% EY (T-EY), (2) AM, or (3) AM with 20% EY (AM-EY). Samples were cooled to 5°C and the following sperm parameters were evaluated after 24 and 48 h of storage: motility, viability, membrane function, acrosome integrity, morphology, and chromatin condensation. Motility was also evaluated after 72 h of storage. The samples that best preserved progressive and total sperm motility at the 24 and 48 h evaluation periods were the ones diluted with AM-EY, observing that with this extender these motility patterns decreased significantly after 72 h of storage compared to time 0 h (p &lt; 0.05). A significant decrease (p &lt; 0.05) in total and progressive motility was observed at 48 h for the T-EY and AM extender compared to 0 h. AM was the only extender in which the percentages of viable sperm decreased significantly (p &lt; 0.05) after 48 h of conservation. For the rest of sperm parameters evaluated, no significant differences were observed between any of the extenders at any evaluation time. The Andromed® extender with the addition of 20% EY could be an alternative option for cooling alpaca sperm obtained from deferent ducts.

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