scholarly journals Effect of warming temperatures on donkey sperm vitrification in 0.5 mL straws in comparison to conventional freezing

2019 ◽  
Vol 17 (3) ◽  
pp. e0406 ◽  
Author(s):  
Maria Diaz-Jimenez ◽  
Jesus Dorado ◽  
Cesar Consuegra ◽  
Blasa Pereira ◽  
Isabel Ortiz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Slow Freezing ◽  
Kinematic Parameters ◽  
Plasma Membrane Integrity ◽  
Progressive Motility ◽  
Acrosome Integrity ◽  
Sperm Freezing ◽  
Conventional Freezing

Aim of study: There is little information about vitrification of sperm in large volumes (up to 0.5 mL). This study aimed to develop the vitrification technique in 0.5 mL straws in donkey sperm, evaluating the effect of three warming temperatures.Area of study: Cordoba, Spain.Material and methods: Ejaculates from five donkeys were divided in four groups: one control subjected to conventional slow freezing (C) and three vitrified in 0.5 mL straws and warmed using different protocols (W1: 37ºC/30s, W2: 43ºC/20s and W3: 70ºC/8s+37ºC/52s). Sperm motility, kinematic parameters, plasma membrane and acrosome integrity were evaluated. Conventional freezing resulted in significantly higher values for total (42.7±19.6%), and progressive motility (30.3±16.7%), plasma membrane (49.1±10.4%) and acrosome integrity (39.6±14.5%) respect to vitrification method.Main results: Values after warming ranged between 0.2-2.8% for total motility; 0.2-2.1% for progressive motility; 5.5-20.0% for plasma membrane integrity and 14.5-29.8% for acrosome integrity in all warming protocols after sperm vitrification. However, no differences were found between W3 and C for kinematic parameters; and W3 resulted in significantly higher values for membrane integrity (20.0±11.0%) in comparison to W1 (5.5±3.6%) and W2 (9.3±8.4%).Research highlights: High warming rates seem to be better for donkey sperm vitrification in large volumes; but this methodology is still not an alternative to conventional sperm freezing.

scholarly journals The Effect of Different Concentrations of Caffeine, Pentoxifylline and 2’-Deoxyadenosine on the Biological Properties of Frozen-Thawed Canine Semen

2019 ◽  
Vol 19 (3) ◽  
pp. 733-746
Author(s):  
Marek Lecewicz ◽  
Rafał Strzeżek ◽  
Anna M. Majewska ◽  
Piotr S. Purpurowicz ◽  
Władysław Kordan
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Artificial Insemination ◽  
Membrane Integrity ◽  
Phosphodiesterase Inhibitors ◽  
Biological Properties ◽  
Kinematic Parameters ◽  
Plasma Membrane Integrity ◽  
Beneficial Effects

AbstractArtificial insemination (AI) and semen cryopreservation are the most accessible and commonly used techniques for breeding domestic animals. Among many parameters, such as plasma membrane integrity and acrosome structure, one of the key factors that determine the quality of frozen-thawed samples for artificial insemination is sperm motility. Sperm motility is one of the key parameters that determine the quality of frozen-thawed samples for AI. The total number of progressively motile spermatozoa in thawed canine semen is correlated with fertility. A variety of substances were used to compare sperm motility with the control. The aim of this study was to determine the effect of semen extender supplementation with motility stimulants, pentoxifylline (PTX), caffeine (CAF) and 2’-deoxyadenosine (DX), after different post-thaw incubation times (30, 60, 120 min) on the motility, selected kinematic parameters, plasma membrane integrity and mitochondrial membrane potential of cryopreserved canine spermatozoa. During attempts to improve the quality of cryopreserved semen, the applied substances exerted beneficial effects at a concentration of 10 mM. We demonstrated that both phosphodiesterase inhibitors, caffeine and pentoxifylline, as well as 2’-deoxyadenosine increased the motility and selected kinematic parameters of thawed canine spermatozoa.

scholarly journals Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

Animals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1462
Author(s):  
Manuel Hidalgo ◽  
Maria Diaz-Jimenez ◽  
César Consuegra ◽  
Blasa Pereira ◽  
Jesús Dorado
Keyword(s):  
Plasma Membrane ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Sperm Motility ◽  
Bovine Serum ◽  
Membrane Integrity ◽  
Plasma Membrane Integrity ◽  
Sperm Suspension ◽  
Direct Exposure ◽  
Conventional Freezing

Vitrification by direct exposure of sperm to liquid nitrogen is increasing in popularity as an alternative to conventional freezing. In this study, the effect of permeable cryoprotectant agents for donkey sperm vitrification was compared to an extender containing non-permeable cryoprotectants. First, three different concentrations of sucrose (0.1, 0.2, and 0.3 molar, M) and bovine serum albumin, BSA (1, 5, and 10%) were compared. Secondly, the concentration of non-permeable agents producing the most desirable results was compared to an extender containing glycerol as permeable agent. Vitrification was performed by dropping 30 μL of sperm suspension directly into LN2 and warming at 42 °C. Sperm motility (total, TM; and progressive, PM) and plasma membrane integrity, PMI (mean ± SEM) were statistically compared between treatments. Sucrose 0.1 M showed a significantly higher percentage of total sperm motility (21.67 ± 9.22%) than sucrose 0.2 M (14.16 ± 4.50%) and 0.3 M (8.58 ± 6.22%); and no differences were found in comparison to the control (19.71 ± 10.16%). Vitrification with sucrose 0.1 M or BSA 5% obtained similar results for TM (21.67 ± 9.22% vs. 19.93 ± 9.93%), PM (13.42 ± 6.85% vs. 12.54 ± 6.37%) and PMI (40.90 ± 13.51% vs. 37.09 ± 14.28); but both showed higher percentages than glycerol (TM = 9.71 ± 4.19%; PM = 5.47 ± 3.17%; PMI = 28.48 ± 15.55%). In conclusion, donkey sperm vitrification in spheres using non-permeable cryoprotectants exhibited better sperm motility and viability parameters after warming than sperm vitrification using extenders containing permeable cryoprotectants.

Optimization of sperm freezability in Bactrian camel using various dilution rates and equilibration times

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 362-366
Author(s):  
Vahid Vahedi ◽  
Mohsen Mostafaei ◽  
Hossein Vaseghi Dodaran ◽  
Nemat Hedayat Evrigh
Keyword(s):  
Plasma Membrane ◽  
Factorial Design ◽  
Healthy Adult ◽  
Membrane Integrity ◽  
Bactrian Camel ◽  
Adult Males ◽  
Kinematic Parameters ◽  
Freezing And Thawing ◽  
Plasma Membrane Integrity ◽  
Progressive Motility

SummaryThe effect of different dilution rates and equilibration times on the cryopreservation of Bactrian camel spermatozoa was evaluated in the current study. Semen samples from four healthy adult males were collected, processed and pooled. They were then subjected to a completely randomized 4×2 factorial design including four dilution rates (DR; 1:1, 1:2, 1:4 or 1:8; v:v with SHOTOR diluent) and two equilibration times (ET; 1 or 2 h at 5ºC). After freezing and thawing, sperm kinematic parameters as well as viability, plasma membrane integrity, abnormality and seminal malondialdehyde level were assessed. According to the results, four-fold diluted samples recorded significantly higher values (P < 0.05) for sperm total (39.58 vs 31.83 and 33.33,%) and progressive motility (19.50 vs 14.00 and 14.25,%), viability (55.37 vs 43.50 and 48.75,%) and plasma membrane integrity (46.75 vs 37.25 and 37.37,%) than those of both less (1:1) and high (1:8) concentrated samples, respectively. By contrast, the percentage of abnormal spermatozoa and the concentration of seminal malondialdehyde were comparable among all treated groups. Moreover, ET revealed that 1 h equilibration had significantly higher sperm motility (37.04 vs 33.33%), linearity (42.29 vs 32.26%), beat cross-frequency (13.15 vs 8.70 Hz), plasma membrane integrity (42.25 vs 39.75%) and viability (51.37 vs 48.12%) compared with 2 h of ET (P < 0.05). Taken together, a four-fold dilution along with 1 h equilibration can be an optimal procedure to cryopreserve Bactrian camel sperm.

scholarly journals Butaphosphan and Cyanocobalamin Supplementation in Semen Extender on Chilled Boar Sperm Quality and Life Span

2020 ◽  
Vol 7 ◽  
Author(s):  
J. Suwimonteerabutr ◽  
S. Chumsri ◽  
P. Tummaruk ◽  
Morakot Nuntapaitoon
Keyword(s):  
Plasma Membrane ◽  
Life Span ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Control Group ◽  
Plasma Membrane Integrity ◽  
Progressive Motility ◽  
Boar Sperm ◽  
Acrosome Integrity ◽  
Semen Extender

The objective of the present study was to determine the effect of butaphosphan and cyanocobalamin supplementation in semen extender on chilled boar sperm quality and life span. A total of 35 ejaculates of boar semen were included. The semen was diluted with Beltsville thawing solution extender supplemented with different concentrations of butaphosphan and cyanocobalamin [0 (control), 0.1, 0.2, 0.3, 0.4, and 0.5%] in the diluted semen. The semen samples were evaluated using a computer-assisted sperm analysis system to determine sperm motility and sperm kinetic parameters (i.e., the curvilinear velocity, VCL; straight line velocity, VSL; average path velocity, VAP; linearity, LIN; straightness, STR; amplitude of lateral head, ALH; wobble, WOB; and beat cross frequency, BCF). Additionally, sperm viability, acrosome integrity, mitochondrial activity, and plasma membrane integrity were evaluated after 4 (day 0), 72 (day 3), 120 (day 5), and 168 (day 7) h of storage using SYBR-14–ethidium homodimer-1 (EthD-1), EthD-1, JC-1, and the short hypo-osmotic swelling test, respectively. The analyses were carried out by using the general linear mixed model (MIXED) procedure of SAS. The statistical models for each data set included group, day after storage, and interaction between group and day after storage. The boar was included as a random effect. On day 0 after storage, progressive motility, VCL, VSL, VAP, and plasma membrane integrity of boar sperm in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the 0.4 and 0.5% groups (P &lt; 0.05). On day 3 after storage, total motility and progressive motility, VCL, VSL, VAP, LIN, WOB, BCF, and plasma membrane integrity in 0.3% of butaphosphan and cyanocobalamin supplementation were significantly greater than those in the control group (P &lt; 0.05). The total motility and progressive motility, VAP, and WOB in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 5 after storage (P &lt; 0.05). No effects of butaphosphan and cyanocobalamin supplementation on acrosome integrity and mitochondria activity were found on days 3, 5, and 7 after storage. However, the motility and progressive motility and the values for all sperm kinetic parameters except ALH in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 7 after storage (P &lt; 0.05). In conclusion, 0.3% of butaphosphan and cyanocobalamin supplementation in semen extender improved sperm motility, sperm activity, morphology, and life span in chilled boar sperm.

50 EFFECTS OF VITAMINS ON THE QUALITY AND FERTILITY OF BOAR SEMEN AFTER LIQUID PRESERVATION AT 5°C

2012 ◽  
Vol 24 (1) ◽  
pp. 137
Author(s):  
Z. Namula ◽  
V. V. Luu ◽  
Y. Kaedei ◽  
R. Kodama ◽  
T. Otoi
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Control Group ◽  
Computer Assisted ◽  
Control Groups ◽  
Plasma Membrane Integrity ◽  
Boar Semen ◽  
Acrosome Integrity ◽  
And Control

Liquid preservation can be used as an alternative to freeze-thawing for preserving semen for AI. The efficiency of some boar semen extenders has been studied over storage periods of 5 to 7 days. The objective of this study was to evaluate the viability and penetrability of boar spermatozoa preserved at 5°C in a modified Modena-based extender supplemented with either 100 μM vitamin C (Vc), 100 μM vitamin E (Ve), or 100 μM Vc + 100 μM Ve (Vc + e). The final sperm concentration was adjusted to cells mL–1 and the semen was then stored at 5°C for 4 weeks. In Experiment 1, the semen samples were assessed every week during the 4-week storage in each extender for the following factors: motility, by using computer-assisted semen analysis (CASA); viability, by using the Live/Dead fluorescence viability assay; plasma membrane integrity, by using the hypoosmotic swelling test (HOST); and acrosome integrity, by using fluorescein isothiocyanate (FITC)-labelled peanut agglutinin staining. In Experiment 2, we examined the penetrability of spermatozoa that had been stored in each extender for 4 weeks and the development of fertilized oocytes. Data were analysed using ANOVA. In Experiment 1, when the semen was stored for 2 weeks, the mean percentage values of total sperm motility and viability for semen stored with Ve were significantly higher than those for semen stored without Vc and Ve (control group) (84.3 vs 67.9% and 59.8 vs 51.2%, respectively; P < 0.05). Moreover, the percentage sperm motility for semen stored for 4 weeks tended to be higher in the Ve group than in the control group (44.2 vs 32.7%; P < 0.1). Storage with Vc or Vc + e did not improve sperm motility and viability of semen. The plasma membrane integrity and acrosome integrity of semen did not significantly differ among the groups during the 4-week storage. In Experiment 2, the rates of sperm penetration and of development to blastocysts of fertilized oocytes did not differ between the Ve and control groups (33.0 vs 28.5% and 14.9 vs 10.1%, respectively; P > 0.05). However, storage with Vc reduced the rate of oocyte development compared with the Ve and control groups (1.1%; P < 0.05). In conclusion, adding Ve to the semen extender may improve the motility and fertility of boar semen stored at 5°C. However, adding Vc has a harmful effect on the quality and fertility of stored boar semen.

Effect of different concentrations of trehalose and glycerol on the freezability of ram semen using soybean lecithin-based diluents

2015 ◽  
Vol 55 (5) ◽  
pp. 666 ◽  
Author(s):  
Zh. Bohlool ◽  
M. Mohammadi ◽  
M. Roostaei-Ali Mehr ◽  
N. Ghavi Hossein-Zadeh
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Soybean Lecithin ◽  
Membrane Integrity ◽  
Sperm Viability ◽  
Plasma Membrane Integrity ◽  
Main Effect ◽  
Acrosome Integrity ◽  
Ram Sperm ◽  
Desirable Effect

This study was conducted to determine the effect of different levels of trehalose and glycerol on ram sperm cryosurvival using lecithin-based diluents. Ejaculates were collected from four rams, pooled after initial evaluation, diluted with Tris-soybean lecithin extender and split into nine equal parts. A total of 0 (T0), 50 (T50) or 100 (T100) mM of trehalose and 3% (G3), 5% (G5) or 7% (G7) of glycerol were added to each part. Sperm motility, viability, plasma membrane integrity and acrosome integrity were evaluated immediately after thawing (0 h), and subsequently after 3 h and 6 h post-thawing incubation at 37°C. Results indicated that there was interaction between trehalose and glycerol on sperm motility. In addition, interaction of trehalose and glycerol with incubation time on sperm motility, viability, plasma membrane integrity and acrosome integrity was not significant (P > 0.05). Sperm motility was greatest in the sperm treated with 100 mM trehalose and 7% glycerol (27%; P < 0.05). The effect of trehalose was significant on viability and plasma membrane integrity of ram spermatozoa (P < 0.05). The main effect of trehalose showed that sperm viability was higher in T100 (47.06%) than T50 (53.96%; P < 0.05). The highest membrane integrity was observed in T100 (47.04%; P < 0.05). Membrane integrity was higher (P < 0.05) in G5 (49.97%) than G3 (41.56%) and there was no difference between G7 (46.86%) and G3 (41.56%; P > 0.05). The best sperm viability and plasma membrane integrity was observed at 0 h (65.75% and 51.58%, respectively). It was concluded that simultaneous use of 7% glycerol and 100 mM trehalose had a desirable effect on motility of ram frozen–thawed sperm.

87 OSMOTIC SENSITIVITY OF OKAPI SPERMATOZOA AND DEVELOPMENT OF CRYOPRESERVATION PROTOCOLS USING CRYOMICROSCOPY

2008 ◽  
Vol 20 (1) ◽  
pp. 124 ◽  
Author(s):  
L. M. Penfold
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Repeated Measures ◽  
Membrane Integrity ◽  
Latin Square ◽  
Crystal Formation ◽  
Plasma Membrane Integrity ◽  
Power Of The Test ◽  
Before And After ◽  
Cooling Effects

Okapi would benefit from artificial insemination with frozen-thawed sperm in cases where aggression prevents mating or where individuals are geographically disparate. Effective sperm cryopreservation is a prerequisite to this goal. Ejaculates (n = 20) were collected from 7 anesthetized adult male okapi housed individually, or with a female for breeding, throughout the year by electroejaculation, and semen and sperm parameters were assessed. Semen aliquots were centrifuged; resuspended in 500 µL of PBS with the osmolarity adjusted to 35, 75, 150, 600, 1200, and 2400 mOsm; and incubated for 30 min before returning to isosmotic conditions. Semen was extended in TEST containing 1%, 2%, or 4% glycerol with or without 0.5% Equex (Minitube, Verona, WI, USA); 5-µL aliquots were cooled in a Latin square design on a Linkam BCS 196 cryomicrostage (Linkam Scientific, Tadworth, Surrey, UK) at 20�C min–1 to –6� –12�C at which point ice crystal formation was induced (seeded), and cooled further to –70�C before warming at 50�C min–1 to 35�C (okapi body temperature). To investigate cooling effects only, raw ejaculate was cooled to –6�C without seeding and warmed to 35�C. Percent sperm motility and plasma membrane integrity (PMI) were recorded before and after treatments. Differences were examined using one-way repeated measures analysis of variance. No differences in motility, total sperm numbers, or percent normal morphology were observed throughout the year (P > 0.05), although the power of the test was low so that negative findings should be interpreted cautiously. Mean semen volume was 1.3 � 0.19 mL, sperm motility was 29 � 3.2%, with a PMI of 39 � 6.8%; 48 � 2.8% were morphologically normal. High proportions of non-motile, plasma membrane-damaged cells were noted in every ejaculate, and whiplash motility, possibly indicating spontaneous capacitation, was observed in several ejaculates 1 h after collection. Motility was dramatically reduced on either side of isosmotic conditions and was more sensitive to osmotic pressure than was plasma membrane integrity. Cooling of raw ejaculate to sub-zero temperatures without freezing did not result in any loss of motility or PMI, indicating cold tolerence. Superior results were obtained when sperm were frozen-thawed in TEST containing 4% glycerol with 0.5% Equex. Findings indicate that okapi semen collected by electroejaculation routinely contain high numbers of non-motile and plasma membrane-damaged spermatozoa, apparently unrelated to season or the length of time since the male was housed with a breeding female. Okapi spermatozoa are remarkably intolerant of departures from isosmotic conditions, indicating a lack of ability to regulate or withstand volume excursions during osmotic stress events; however, cooling to sub-zero temperatures in the absence of cryoprotectant did not reduce percent sperm motility or PMI, indicating resistance to cold shock. Increasing and maintaining proportions of motile, membrane-intact spermatozoa prior to and during cryopreservation will be critical for development of freezing protocols for this species.

Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function

2019 ◽  
Vol 31 (9) ◽  
pp. 1434
Author(s):  
Andressa Dalmazzo ◽  
João D. A. Losano ◽  
Daniel S. R. Angrimani ◽  
Isabel V. A. Pereira ◽  
Marcelo D. Goissis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Expression ◽  
Membrane Integrity ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Mitochondrial Activity ◽  
Hormone Binding ◽  
Plasma Membrane Integrity ◽  
Gene And Protein Expression ◽  
Acrosome Integrity

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

The effects of chronic administration of ghrelin on rat sperm quality and membrane integrity

2009 ◽  
Vol 59 (2) ◽  
pp. 159-168 ◽  
Author(s):  
Arash Kheradmand ◽  
Majid Taati ◽  
Homayoon Babaei
Keyword(s):  
Plasma Membrane ◽  
Treatment Group ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Chronic Administration ◽  
Control Group ◽  
Plasma Membrane Integrity ◽  
Significant Difference

AbstractAlthough ghrelin acts as a modulator of feeding behavior and energy metabolism in the central nervous system, recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we investigated the effects of chronic administration of ghrelin on the motility, plasma membrane integrity and concentration of rat spermatozoa. 45-d male Wistar rats were scheduled for the study and were divided into control and treatment groups. In the treatment group, 1 nmol of ghrelin was administered as sc injection for 10 consecutive days or vehicle (physiological saline) to the control rats. Sperm collection was achieved by killing of the rats on days 15, 25 and 50 after first injection. Total sperm motility and forward progressive movement did not exhibit significant difference during the experiment, although, there was a tendency for greater motion rate on d 15 and 25 in the treated rats compared to the control group. Plasma membrane integrity (HOS-reacted spermatozoa) was significantly higher in the treated animals, especially on day 15 as well as day 25, because of possible antioxidant properties of ghrelin. This value was statistically higher on day 15 than that of day 25 (P <0.05). Likewise, there was a significant correlation between the FPM (P <0.0001, r = 0.79) and TSM (P <0.01, r = 0.52) with the HOS test percentage in the treatment group. It was not observed statistically difference in the sperm concentration between groups during all of the experimental days. In conclusion, chronic administration of ghrelin (similar to induced by energy deficiency such as fasting) increased the integrity of sperm membrane, however, the sperm motility and concentration did not display any alterations.

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