scholarly journals 79 EFFECT OF PRE-FREEZE ADDITION OF PLATELET-ACTIVATING FACTOR AND PLATELET-ACTIVATING FACTOR:ACETYLHYDROLASE ON THE POST-THAW INTEGRITY OF FROZEN - THAWED BOAR SPERM

2005 ◽  
Vol 17 (2) ◽  
pp. 189
Author(s):  
R. Bathgate ◽  
B.M. Eriksson ◽  
W.M.C. Maxwell ◽  
G. Evans

The use of frozen-thawed boar sperm is not widespread, owing to reduced fertility rates and high cost per dose (Eriksson et al. 2004 Proc. Aust. Assoc. Pig Vet., 61–69). Improvements in post-thaw sperm survival are required for commercialization. Platelet-activating factor (PAF) is a phospholipid involved in regulating sperm function. PAF:acetylhydrolase (PAF:AH) regulates PAF activity by conversion to its inactive isoform. Both occur naturally in boar semen (Kordan et al. 2003 Pol. J. Vet. Sci. 6, 55–60). Removal of PAF and PAF:AH along with seminal plasma during the cryopreservation process may inhibit the ability of sperm to withstand the freeze-thawing process. The aim of this study was to assess the effect of PAF and PAF:AH, added to boar semen pre-freeze, on the post-thaw motility and acrosome integrity of sperm. The sperm rich fraction was collected from a mature Large White × Landrace boar, diluted with Androhep (1:2, semen:Androhep; Minitube, Verona, WI, USA), cooled to 17°C over 2 h, and then centrifuged (10 min, 800g). The sperm pellet was resuspended in cooling extender (11% (w/v) lactose solution with 20% (v/v) egg yolk; control), cooling extender plus 100 ng/mL PAF (PAF), or cooling extender plus 0.4% (v/v) PAF:AH (Pafase; ICOS Corporation, Seattle, Washington, USA), and cooled to 5°C over 2.5 h. Sperm were further diluted with cooling extender plus 9% (v/v) glycerol and 1.5% (v/v) Equex STM (freezing extender), loaded into 0.5-mL straws, and frozen. Straws were thawed (20 s, 42°C) and the motility and acrosome integrity (FITC-PNA; Mortimer etal. 1990 Hum. Reprod. 5, 99–103) assessed at 0, 3, and 6 h post-thaw after incubation at 37°C. Data from three replicates were analyzed by ANOVA and a Tukey test applied where significant differences were found. Post-thaw motility (0 and 3 h) was higher for PAF (60.0 ± 0.0% and 25.0 ± 2.9%) than for control (41.7 ± 1.7% and 10.0 ± 2.9%; P < 0.05), but was similar for Pafase (41.7 ± 1.7% and 16.7 ± 1.7%; P > 0.05). By 6 h post-thaw, motility was similar for PAF (1.7 ± 1.7%), Pafase (6.7 ± 6.8%), and control (1.7 ± 1.7%, all respectively; P > 0.05). Acrosome integrity was higher at 0, 3 and 6 h post-thaw for Pafase (55.7 ± 3.2%, 45.7 ± 3.7% and 23.0 ± 3.1%) than for control (42.7 ± 1.5%, 25.7 ± 5.7% and 12.3 ± 2.7%) and PAF (33.0 ± 3.7%, 26.3 ± 2.2% and 11.7 ± 0.3%, all respectively; P < 0.05), but was similar between control and PAF (P > 0.05). Supplementation of cooling extender with 100 ng/mL PAF increased initial post-thaw motility, but this benefit was lost after 6 h post-thaw. Pafase in the cooling extender improved the proportion of intact acrosomes, even after 6 h post-thaw. In vitro studies investigating the interaction between Pafase-treated frozen-thawed sperm and oviducal epithelial cells would be of interest to further establish the potential benefits of pre-freeze addition of Pafase on the fertilizing potential of frozen-thawed boar sperm.

2002 ◽  
Vol 14 (8) ◽  
pp. 509 ◽  
Author(s):  
Li-Jun Huo ◽  
Kui-Zhong Yue ◽  
Zeng-Ming Yang

Extended storage of unfrozen boar semen becomes an alternative because the use of frozen–thawed boar sperm results in low fertility. Sperm viability, mitochondrial activity, capacitation and acrosome integrity of freshly ejaculated boar semen stored in vitro for up to 48 h at 4°C, 15°C, 20°C and 39°C was characterized during the study. The viability of boar sperm was assessed by both Hoechst 33258 and SYBR-14/PI staining. Mitochondrial function was assessed by JC-1 staining. Capacitation status was determined by chlortetracycline (CTC)/Hoechst 33258 staining. The acrosome integrity was analysed with Coomassie blue staining. These data were derived from three ejaculates each from three crossbred boars. The viabilities assessed with SYBR-14/PI, Hoechst 33258 and JC-1 staining correlated highly (r > 0.980). In freshly ejaculated boar semen, 96 ± 1% of the sperm did not take up the Hoechst 33258, whereas 95 ± 2% were stained by SYBR-14 and 96 ± 2% of the sperm had mitochondria exhibiting positive JC-1 staining. Staining with CTC/Hoechst 33258 suggested that a high percentage of sperm became capacitated after 24 h storage at 15°C and 20°C. There were 62 ± 2% (15°C) and 89�±�2% (20°C) capacitated sperm by 48 h. Moreover, most of the capacitated sperm were acrosome intact. These results suggest that SYBR-14/PI, Hoechst 33258 or JC-1 staining can be used to effectively evaluate the quality of boar sperm during in vitro storage.


2005 ◽  
Vol 17 (2) ◽  
pp. 195
Author(s):  
C. Matas ◽  
J. Gadea ◽  
G. Decuadro-Hansen

Boar semen freezing procedures include the use of centrifugation to concentrate sperm and remove seminal plasma prior to dilution in freezing extender. The centrifugation techniques employed have necessarily been a compromise between the need to recover as many spermatozoa as possible after centrifugation and the damage caused by pelleting the sperm. The use of an inert, dense, and isotonic solution as a cushion in the bottom of the tube leads to the use of higher-speed centrifugation to ensure maximum sperm recovery. However, it is necessary to know the viability and functionality of the samples after the thawing process. The aim of this work was to evaluate the effect of cushion-technique centrifugation on the in vitro sperm viability and the penetrating capacity after thawing. Sperm-rich fractions from five fertile boars were diluted and cooled to 15°C before centrifugation. Two centrifugation regimes were used: 800g for 10 min called the “standard method” (SM) (Westendorf P etal. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267) and 1000g for 20 min on an iodixanol isotonic solution 60% w/v gradient (Sigma Chemical Co., St. Louis, MO, USA) called the “cushion method” (CM). Spermatozoa were diluted in lactose/egg-yolk extender, cooled to 5°C over 2 h and then frozen with glycerol and Equex by classic methodology (Westendorf P et al. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267). Frozen sperm samples were thawed in a circulating water bath at 38°C for 30 s. To detect increases in plasma membrane lipid packing disorder and viability, frozen-thawed samples of sperm were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378–391) and evaluated by flow cytometry. In vitro penetration ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Gadea and Matas 2000 Theriogenology 54, 1343–1357). ANOVA analysis revealed that centrifugation by CM showed higher values of intact viable spermatozoa than SM centrifugation (60.21 v. 54.68%, P < 0.05). The in vitro penetration assay showed no differences in penetration rate or mean number of sperm penetrated per oocyte. However, significant boar and interaction effects were found (P < 0.01). These results indicated that different effects of the treatment were found for every boar. In conclusion, the cushioned centrifugation method gives a simple means of processing porcine semen for freezing more efficiently without loss of fertilizing capacity. This work was supported by AGL-2003-03144.


2013 ◽  
Vol 25 (1) ◽  
pp. 176
Author(s):  
R. Athurupana ◽  
H. Funahashi

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The use of glycerol for boar semen cryopreservation may be a reason of low fertility results. Trehalose is a nonreducing disaccharide known to stabilize proteins and biologic membranes during processes such as cryopreservation. The aim of this study was to evaluate the effects of trehalose on boar sperm cryosurvival in an egg-yolk-based extender. The semen samples collected from different individual Berkshires were diluted in egg-yolk-based freezing extender containing glycerol (final concentration 68.5 and 274 mM) or trehalose (50 and 100 mM). Then the samples were cryopreserved using the straw freezing procedure. Frozen sperms were thawed at 39°C in water. Post-thawed sperm were analyzed for motility (under microscope by a conventional method), viability, and acrosome integrity (under fluorescence microscope following LIVE/DEAD or CTC staining, respectively). Statistical analyses of results from 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post hoc test (significance; P < 0.05). The extender supplemented with 100 mM trehalose exhibited significantly higher acrosomal integrity (41.6%) compared with other extenders (P < 0.05, n = 5; Table 1). The trend of motility and viability was higher in 274 mM glycerol (28.0 and 52.9%) and 100 mM trehalose extenders (23.6 and 53.9%), but those were not significant. These results demonstrate that the presence of trehalose at 100 mM during cryopreservation improves the acrosome integrity of boar sperm, without any reduction in viability and motility, after thawing. Table 1.Effect of glycerol and trehalose on post-thaw boar sperm


2021 ◽  
Vol 41 (1) ◽  
pp. 16-27
Author(s):  
J. O. Daramola ◽  
T. A. Sorongbe ◽  
O. M. Onagbesan ◽  
A. V. Jegede ◽  
A. O. Ladokun ◽  
...  

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.


1987 ◽  
Author(s):  
Cs Perger ◽  
A von Felten

PAF is suggested to be of pathophysiological importance in a variety of diseases. Since platelets exhibit a reduced sensitivity to PAF after a contact with this agent, this behavior may be used as indicator of PAF released into the circulation. In extrinsic asthma, platelets show a diminished reaction to PAF after exposition of the patients to the antigen compared to their own platelets before exposition (Beer and von Felten, Adv. Inflamm. Res. 10:323,1986). We were therefore looking for a test system indicating directly whether platelets had been in contact with PAF.Preparation of PAF-desensitized platelets: Citrated PRP was placed in a cuvette of an aggregometer, and PAF was added in 10 portions at intervals of 10 sec (37oc, constant stirring) to a final concentration of 10 to 100 nM, depending on the individual sensitivity of each platelet preparation. Therby, only a minimal, completely reversible aggregation was registered without any release of serotonin (ST) or 3-thromboglobulin (BTG). Control platelets were pretreated with buffer instead of PAF. Both platelets preparations were kept at 37°C for 45 min. Whereas control platelets showed a secondary aggregation to PAF (5x conc. used for desensitization), PAF-pretreated piatelets were only reversibly aggregated.Sensitivity of PAF-desensitized and control platelets to other platelet agonists: No difference in aggregation, ST-or BTG-relea-se was observed after stimulation with several concentrations of ADP, collagen and arachidonate (p>0.05,n= 41).Binding of 3H-PAF to platelets: PAF-desensitized and control platelets were separated from plasma by filtration through sepharose CL-2B (Pharmacia) in hepes-buffered Tyrode’s solution. After incubation with 3H-PAF, platelets were washed on Whatman 934-AH filters (vacuum filtration). On desensitized and control platelets, we found 175±48 (mean±sd) and 231±70 3H-PAF molecules / platelet respectively after incubation with 5 nM ^h-PAF, 399±36 and 504±66 ^H-PAF molecules / platelet after incubation with 20 nM. In spite of a statistically significant reduction of PAF-binding after desensitization (p<0.01),the variability of PAF-binding between platelets of different individuals is too high to allow a discrimination of normal from PAF-desensitized platelets.


2009 ◽  
Vol 21 (1) ◽  
pp. 138
Author(s):  
J. E. Rodríguez-Gil ◽  
M. Hernández ◽  
M. M. Rivera ◽  
L. Ramió-Lluch ◽  
J. Ballester ◽  
...  

The optimization of freezing extenders is an essential issue for enhancing boar sperm cryosurvival. The aim of the present study was to disclose the role of glucose concentration of freezing extender on the metabolic activity of frozen–thawed spermatozoa. To achieve it, pooled sperm-rich ejaculate fractions from 5 mature and fertile boars (3 ejaculates per boar) were collected using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet was split into 7 aliquots. The aliquots were diluted to a final concentration of 1 × 109 sperm mL–1, in a Tris-citric extender supplemented with 20% egg-yolk, 3% glycerol, and 0, 0.05, 2, 4, 10, 55, or 185 mm glucose. All the extenders were adjusted to a pH of 6.8 and 310 mOsm kg–1 to avoid osmolarity effects. Extended semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20°C min–1. Thawing was carried out in a water bath at 37°C for 20 s. Afterward, an analysis of protein phosphorylation in tyrosine residues was carried out through bi-dimensional electrophoresis followed by a Western blot analysis. This analysis indicated that sperm samples frozen in extenders without glucose showed specific changes in the tyrosine phosphorylation pattern compared with fresh sperm. Furthermore, the addition of glucose in increasing concentrations to the freezing extender was accompanied by a concentration-dependent decrease in the overall tyrosine phosphorylation pattern, especially in proteins with a molecular weight ranging from 150 to 200 kDa and an acidic isoelectric point (pI). The maximal decrease was observed in spermatozoa frozen in the extender containing 185 mm glucose, in which an additional decrease in the tyrosine phosphorylation of proteins ranging from 60 to 80 kDa, and a basic pI was also observed. These results suggest that glucose is a modulator in the resistance of boar sperm to support freezing and thawing process, because the precise protein phosphorylation pattern of spermatozoa is directly linked to their functional status. In this way, a precise control of the glucose concentration of the freezing extender would be required to improve boar sperm cryoresistance. Supported by CICYT (AGL2005-00760 and AGL2004-04756-C02-02/GAN), Madrid and GERM (04543/07), Murcia, Spain.


1972 ◽  
Vol 52 (1) ◽  
pp. 65-72 ◽  
Author(s):  
L. M. SANFORD ◽  
G. J. KING ◽  
J. W. MACPHERSON

Boar and bull spermatozoa were diluted in a skim milk–egg yolk–glucose extender containing 0, 7.5, or 15% glycerol (v/v) and incubated aerobically for 6 hr at 37 C. Other partially diluted boar semen samples were cooled to 5 C. Glycerol was added to a final concentration of 0, 7.5, and 15%. Samples were frozen to −79 C, rewarmed, and incubated for 3 hr at 37 C. The presence of glycerol in the extender depressed (P < 0.01) the oxygen uptake by nonfrozen boar and bull spermatozoa during the 6-hr incubation period. The reduction of oxygen uptake by semen samples increased as the level of glycerol in the extender increased. There was a corresponding decrease (P < 0.01) in the number of motile cells at the conclusion of the incubation period. Glycerol appeared to have more of a detrimental effect on boar spermatozoan oxygen uptake. The rate of oxygen uptake by boar semen samples postfreezing was extremely depressed, suggesting that spermatozoa surviving the freezing process metabolize at a much lower rate than normal. Active progressive motility of most of the surviving boar spermatozoa ceased within 1–2 hr of incubation under the in vitro conditions of this experiment.


2005 ◽  
Vol 17 (2) ◽  
pp. 305
Author(s):  
I. Parrilla ◽  
J.M. Vazquez ◽  
M.A. Gil ◽  
I. Caballero ◽  
C. Almiñana ◽  
...  

Addition of seminal plasma (SP) to the collection medium has been shown to be beneficial for motility and viability of sex-sorted and stored spermatozoa. However, SP could not only delay but also decrease the in vitro fertilization rates of IVM pig oocytes. In the present study, the time-course of IVM pig oocyte penetration of sex sorted boar spermatozoa stored in the presence or absence of SP was evaluated. Spermatozoa were sex-sorted following the Beltsville sperm sexing technology (Johnson and Welch 1999 Theriogenology 52, 1323–1342) and collected in TEST-egg yolk buffer (2%) with (10%) or without (control) SP. Sex-sorted spermatozoa were stored at 20°C during 0, 2, 5, and 10 h after sorting. Oocytes were matured in vitro in NCSU23 (Peters and Wells 1663 J. Reprod. Fertil. 48, 61–73) for 44 h in 5% CO2 in air at 39°C. The in vitro penetration time-course was determined by co-incubating the sex-sorted and stored spermatozoa with IVM oocytes during 3, 6, and 18 h in modified TRIS-buffered medium (mTBM) (Abeydeera and Day 1997 Theriogenology 48, 537–544) at 39°C in an atmosphere of 5% CO2 in air. Penetration rates and number of spermatozoa per oocyte were assessed after fixation and staining of the oocytes. Statistical analyses were conducted by ANOVA. Presence of SP did not delay the onset of the oocyte penetration. Moreover, at 3 h of co-incubation, SP increased (P < 0.05) both penetration rates and mean number of spermatozoa per oocyte in sorted and stored boar spermatozoa when compared with control (45 vs. 20, 50 vs. 32, 38 vs. 23, 15 vs. 8, at 0, 2, 5, and 10 h of storage with SP and control, respectively). High penetration rates were reached after 6 h of co-incubation (82 vs. 51, 96 vs. 76, 83 vs. 48, 31 vs. 24, at 0, 2, 5, and 10 h of storage with SP and control, respectively) in sorted and stored samples, with no further increase at 18 h (70 vs. 63, 92 vs. 79, 87 vs. 53, 55 vs. 40, at 0, 2, 5, and 10 h of storage with SP and control, respectively). Spermatozoa stored 2 h in the presence of SP showed the best penetration rate and highest mean number of spermatozoa per oocyte. The mean number of spermatozoa per oocyte increased as the co-incubation time increased (ranging from 2.1 to 5.8 for sorted spermatozoa stored 2 h in the presence of SP at 3 h and 18 h of co-incubation, respectively). In conclusion, the presence of SP during the storage of sex-sorted spermatozoa improves their in vitro fertilizing ability without affecting the onset of the oocyte penetration time. This work was supported by DGICYT (AGL 2001-0471), Fundación Seneca (PB174/FS/02) and CTIC (2103SIU0040).


Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 175-181 ◽  
Author(s):  
Peng Wang ◽  
Yan-Feng Wang ◽  
Chun-Wei Wang ◽  
Shu-Hai Bu ◽  
Jian-Hong Hu ◽  
...  

SummaryLow-density lipoproteins (LDL) is known to protect boar sperm during freezing–thawing, but little information is known about the effects of LDL extracted from different avian egg yolks on post-thaw boar semen quality. The purpose of this study was to compare and analyze the effects of LDL at various concentrations and different species on boar sperm quality after freezing–thawing. LDL extracted from the yolk of hen egg, duck egg, quail egg, pigeon egg or ostrich egg was added to the extender at the concentrations of 0.06, 0.07, 0.08, 0.09 and 0.1 g/ml, respectively, and their effects on frozen–thawed boar sperm quality were assessed. According to all measured parameters, the results showed that sperm motility, acrosome integrity and plasma membrane integrity were 43.20%, 52.57% and 48.13%, respectively, after being frozen–thawed with 0.09 g/ml LDL extracted from pigeon egg yolk. All these quality parameters were higher than that of other groups (P < 0.05). In conclusion, our results confirmed that LDL extracted from pigeon egg yolk had the best cryoprotective effects on frozen–thawed boar sperm among all of the groups supplemented with LDL from five kinds of avian egg in extender. The optimum concentration of LDL extracted from pigeon egg in boar semen freezing extender was 0.09 g/ml.


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