Boar Sperm Quality Research Articles

Improvement of the boar sperm quality by including of spirulina platensis in the diet

Improvement of the boar sperm quality by including of spirulina platensis in the diet

Polymyxin B neutralizes bacteria-released endotoxin and improves the quality of boar sperm during liquid storage and cryopreservation

Gram negative bacteria are the predominant type detected in boar semen. Since these bacteria release lipopolysaccharide (LPS), and because polymyxin B (PMB) neutralizes LPS activity, the objective was to improve techniques for long-term storage of boar sperm by testing the beneficial effects of PMB. In the present study, LPS bound directly to the head region of sperm, decreased sperm motility, and induced sperm apoptosis. The addition of 100 μg/mL PMB suppressed LPS binding activity and blocked the negative effects of LPS on sperm quality. Additionally, when PMB treatment was combined with penicillin G (PenG), sperm motility was increased after 10 d of liquid storage or in frozen-thawed sperm (P<0.05). When the PMB- and PenG-treated sperm was used for artificial insemination, the conception rate was increased relative to that of artificial insemination with sperm treated by PenG alone in both liquid (62 vs. 81%) and cryopreserved forms (50 vs. 80%, P < 0.05). We concluded that PMB suppressed LPS-induced low sperm motility and apoptosis via the reduction of LPS binding to Toll-like receptors (TLRs). Thus, in order to enhance sperm quality for artificial insemination, a combined treatment with PMB and PenG immediately after ejaculation seemed appropriate to maintain sperm motility and function during both liquid storage and cryopreservation.

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility.

EFFECT OF SEASON ON BOAR SPERM MORPHOLOGY

The objective of this study was to analyze the influence of the year–season effect on semen production parameters in the fertile AI boars. The evaluation was especially focused on the morphologically abnormal spermatozoa (MAS) incidence. It was microscopically evaluated after making fresh semen smears and staining on microscopic slides. MAS incidence 19.46 % was lower at first half-year than at second half-year 25.00 % (P<0.01). Spermatozoa with distal protoplasmic droplet were furthest participated in total MAS incidence. Its rate was the highest at fourth quarter in comparison with annual period (P<0.001). We found that season has the negative effect on sperm morphology and significantly affects boar sperm quality and subsequently AI dose quality.

The Cryoprotective Effect of Trehalose Supplementation on Boar Spermatozoa Quality

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200 mm), and try to determine the optimum concentration of trehalose. We chose sperm motility, mitochondrial activity, acrosome integrity and membrane integrity as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100 mm trehalose-supplemented extenders, with values of 49.89% for motility, 44.69% for mitochondrial activity, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance diminished significantly for 200 . The synergic effect of trehalose and glycerol resulted in better cryosurvival of boar spermatozoa than that of a single cryoprotectant. In conclusion, when trehalose-supplementation was added up to 100 mm, trehalose confers a greater cryoprotective capacity to the extender, and the sperm motility, mitochondrial activity, membrane integrity and acrosome integrity parameters were significantly improved during frozen-thawed process.

Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen–thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5mg/ml GPP, as compared to other groups (P<0.05). The extender supplemented with 0.5mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P<0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0mg/ml GPP than that of other treatments, and the control group (P<0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5mg/ml GPP significantly improved SOD levels, compared to other groups (P>0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P>0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen–thawed boar semen. It is proposed that an extender containing 0.5mg/ml GPP could be used as cryoprotective medium of better efficiency.

Effects of Straw Volume and Equex‐STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN(2)) vapour approximately 3 cm above the level of LN(2) for 20 min and then were plunged into LN(2). Thawing was achieved in warm water at 50 degrees C for 12 s and then was incubated in a 38 degrees C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM was added to the freezing extender. There was no difference (p = 0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p = 0.02, p = 0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM (p > 0.05). The results of these investigations suggested that Equex-STM exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.

Effect of the spirulina platensis included in the main diet on the boar sperm quality

Microalgae Spirulina platensis accumulates many chemical components which are suitable for all higher organisms as food and forage raw material. There are a lot of vitally important for the organisms minerals and macroelements such as iron, calcium, sodium, potassium, copper, magnesium, phosphorus, selenium, vitamins, carotin, nucleic acids, enzymes and other active substances. That should explain the value of Spirulina as a feed additive for the agricultural animals. In the present work the influence of the microalgae Spirulina platensis on the quantitative and qualitative characteristics of boars' sperm was studied. The experiment was carried out with 6 boars from Danube white breed in the experimental animal base of the Agricultural Institute - Shumen. The time of the experiment was divided in the control and experimental periods. During the control period the animals received the main diet in accordance with Bulgarian state standard BDS- 1642-96. In the experimental period to the main diet was added 7 ml/ per head the fresh biomass of microalgae preserved by melasa (final quantity - 1,4 mg Spirulina platensis). The obtained results shown that the addition of Spirulina improves the sperm parameters in boars: the volume of the ejaculates increased with 30 ml in experimental period (306 ml against 276 in control, P&lt;0,05) as well as the spermatozoa concentration enhanced with 27 mln/ml. The total dehydrogenases activities estimated by reaction with methylene blue as well as LDH activity were higher in the experimental period. Also the survivability of the spermatozoids at 24, 48 and 72 hour in the experimental period was more than in control.

Boar sperm quality after co-culture with homologous oviductal epithelial cells

Boar sperm quality after co-culture with homologous oviductal epithelial cells

Ubiquitination and its influence in boar sperm physiology and cryopreservation

Recent reports document the potential use of the ubiquitin protein as an indicator of mammalian sperm quality or fertility, based on poor morphology, sperm count, and other cellular qualities. However, its influence on cellular physiologic mechanisms and boar sperm cryopreservation are unknown. The objective of this research was to determine the influence of boar sperm ubiquitination ( n = 12 boars) on motility (using CASA), and flow cytometry and fluorescent probes (in parentheses) to evaluate mitochondrial activity (JC-1), plasma and acrosomal membrane integrity (PI and FITC-PNA), membrane fluidity (M540), and chromatin stability (TUNEL) for fresh and frozen–thawed samples. The effects of ubiquitination (determined flow cytometrically) on the ability of frozen–thawed boar sperm to capacitate (FLUO-3AM) and acrosome react (FITC-PNA) were also investigated using flow cytometry. Cryopreservation induced a decrease in the percentage of sperm that were ubiquitinated from 29 to 20% ( P < 0.0001), but no significant effects of ubiquitin on sperm quality (motility, membrane integrities and organization) were detected. The ability of sperm to capacitate and acrosome react was influenced by ubiquitination. Samples with more ubiquitinated boar sperm were able to maintain plasma membrane integrity (PMI) better and have fewer live acrosome-reacted cells over 120 min of induced capacitation ( P < 0.05). In conclusion, frozen–thawed ubiquitinated boar sperm were better able to survive the physical stresses of induced capacitation, yet were still capable of capacitating and acrosome reacting, which may enable use of this assay for in the vitro evaluation of the quality of boar sperm.

Effect of liquid storage on membrane integrity and mitochondrial activity: a new diagnostic method of evaluating boar sperm quality

Effect of liquid storage on membrane integrity and mitochondrial activity: a new diagnostic method of evaluating boar sperm quality

RETRACTED: The cryoprotective effect of trehalose supplementation on boar spermatozoa quality

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200mmol/l), and tried to determine the optimum concentration of trehalose. We chose sperm motility, acrosome integrity, membrane integrity and cryocapacitation as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100mmol/l trehalose-supplemented extenders, with values of 49.89% for motility, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance was diminished significantly for 200mmol/l of trehalose. Before and after capacitation, the CTC score for semen diluted by extender containing 100mmol/l trehalose was 3.68% and 43.82%, respectively. In conclusion, trehalose could confer a greater cryoprotective capacity to boar spermatozoa. Trehalose-supplementation with 100mmol/l concentration in basic extender could significantly improve sperm motility, membrane integrity and acrosome integrity parameters, and reduce boar spermatozoa cryocapacitation during the cryopreservation process.

Boar spermatozoa and prostaglandin F 2α: Quality of boar sperm after the addition of prostaglandin F 2α to the short-term extender over cooling time

Prostaglandin F 2α (PGF 2α) has been used to improve reproductive performance in swine. The goal of the present work was to determine how the addition of PGF 2α affects boar sperm quality. Eleven different treatments were evaluated: eight with only PGF 2α (0.625, 1.25, 2.50, 5, 10, 12.50, 25 and 50 mg PGF 2α/100 ml) and three binary treatments (0.625 mg PGF 2α/100 ml + 200 μg/ml hyaluronic acid (HA), 1.25 mg PGF 2α/100 ml + 200 μg/ml HA, 0.625 mg PGF 2α/100 ml + 7.5 μM caffeine (Caf)). All these substances were added to 16 ejaculates from 16 healthy and sexually mature boars ( n = 16), and each ejaculate was considered as a replicate. Our study also assessed the effects of these 11 treatments over different periods of preservation. Sperm quality was tested immediately after the addition of treatments (time 0), and after 1, 3, 6 and 10 days of cooling at 15 °C. To evaluate sperm quality, five parameters were analysed: (1) sperm viability, acrosome and mitochondrial sheath integrity (using a multiple fluorochrome-staining test), (2) sperm motility, (3) sperm morphology and (4) agglutination (using a computer assisted system) and (5) osmotic resistance (using the ORT). Parametric (analysis of variance for repeated measures) and non-parametric tests (Friedman test) were used as statistical analyses. Treatments with PGF 2α concentrations higher than 12.5 mg/100 ml were cytotoxic while the others did not damage boar spermatozoa. Thus, the other treatments may be used to produce profitable effects without adverse effects. Moreover, the addition of PGF 2α at 5 mg/100 ml to sperm diluted in BTS may maintain sperm viability and motility better after 6 days of cooling, because significant differences were observed ( P < 0.05) compared with control at the same time.

Dialysis of boar semen prior to freezing–thawing: Its effects on post-thaw sperm characteristics

Low-molecular weight components of the seminal plasma have a detrimental effect on sperm function. The present study was undertaken to evaluate the effect of the removal of low-molecular weight components by dialysis on sperm characteristics prior to and after freezing. Semen, collected from 5 boars, was extended in Kortowo-3 extender (K-3, Poland) and cooled for 3 h (control non-dialysis) or dialyzed for 5 h in semi-permeable dialysis bags of 12–14 kDa molecular weight cut-off prior to freezing. The semen samples were diluted in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or lactose-lyophilized lipoprotein fractions-glycerol extender (lactose-LPFo-G), packaged into aluminum tubes and frozen in a controlled programmable freezer. Pre-frozen and frozen–thawed spermatozoa were evaluated for motility, plasma membrane (SYBR-14 and propidium iodide) and acrosome integrity, mitochondrial function (Rhodamine 123) and ATP content. The results of the study showed that dialysis significantly improved the sperm characteristics prior to freezing. Dialysis enhanced ( P < 0.05) post-thaw sperm motility, plasma membrane integrity and mitochondrial function, but had no significant effect ( P > 0.05) on recovery of spermatozoa with intact acrosomes. Furthermore, dialyzed spermatozoa exhibited higher ( P < 0.05) ATP content compared with the control after freezing–thawing. Consistent inter-boar variability was detected mainly in dialyzed semen following freezing–thawing. These results indicated that the improvement in sperm quality characteristics prior to freezing and the post-thaw sperm recovery were due to the removal of low-molecular weight components from the seminal plasma. It can be suggested that dialysis is effective in improving the post-thaw quality of boar spermatozoa and has also great practical importance in improving the protocols for cryopreservation of semen. Dialysis may also contribute to a better understanding of different mechanisms underlying cryo-induced damage to boar spermatozoa.

Improvement of Sperm Production in Subfertile Boars byCordyceps militarisSupplement

Cordyceps species have been traditionally used for the enhancement of sexual function, however, there is few direct evidence to prove this. We investigated the spermatogenic effect of Cordyceps militaris (CM) by supplementation with CM mycelium to subfertile boars. Seventeen Duroc and 12 Landrace boars (29 to 40 months old) were selected to feed with regular diet (control groups, n = 8 and 6, respectively) or diet supplemented with CM mycelium (treatment groups, n = 9 and 6, respectively) for 2 months. Semen was collected once a week. The quality of fertile sperm (normally greater than 62% of motility and 70% of normal morphology) and the quantity (semen volume, and total sperm number) were compared in these boars. The result showed that sperm production was enhanced significantly at the end of first month (p < 0.05), peaked at the second month (p < 0.01) of supplementation with CM and was maintained for 2 weeks after stopping the treatment (p < 0.01). Plasma cordycepin concentration was detected in boars supplemented with CM but not in the controls. More importantly, the percentages of motile sperm cells and sperm morphology were also improved significantly in most of treated boars during the second month of supplementation (p < 0.01) and 2 weeks after the treatment (p < 0.05) as compared to their initial values. These results indicate that supplementation with CM mycelium improves sperm quality and quantity in subfertile boars and may partly support the role of Cordyceps in sexual enhancement.

Does exposure to di(2‐ethylhexyl) phthalate in pre‐pubertal boars affect semen quality post‐puberty?

Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinyl chloride (PVC) products, has been ascribed to have toxic effects on animal reproduction. The present study aimed at determining potential late effects of pre-pubertal oral exposure to DEHP on semen quality in young pigs. Ten pairs of cross-bred male siblings were used. One brother in each pair became, at random, the test animal while the other acted as control. Test males were exposed to 300 mg/kg body weight (bw) of DEHP administered orally three times a week from 3 to 7 weeks of age. The control group was given placebo (water). Semen analyses started when the boars reached 6 months of age, with semen collected twice weekly, until animals were 9 months of age. Semen was evaluated for ejaculate volume, sperm concentration, total sperm count, sperm motility, sperm morphology (including presence of cells other than spermatozoa) and sperm plasma membrane integrity. Total sperm motility tended to be lower while local motility was higher in the DEHP-exposed group compared with controls (p = 0.07) when assessed by computer-assisted sperm analysis. The DEHP-exposed group had a significantly (p < 0.05) lower percentage of spermatozoa with tailless, defective heads (at 7-8 months of age) and double-folded tails (at 6-7, 7-8 and 6-9 months of age), compared with controls (albeit always under 5%). In summary, there were no obvious adverse effects of early oral exposure to 300 mg/kg bw of DEHP on sperm output and sperm quality in post-pubertal young boars.

Detection of early changes in sperm membrane integrity pre-freezing can estimate post-thaw quality of boar spermatozoa

A recently developed triple staining (SNARF-1/YO-PRO-1/ethidium homodimer) was used to assess early changes in boar sperm membrane integrity (MI) with the results of cryopreservation procedures and to seek for correlations among MI-spermatozoa in pre-freeze semen and its freezeability. Ejaculates from five boars were evaluated in the fresh and frozen-thawed (FT) state, and its freezeability defined as % of membrane intactness, MI% (MI% = % of FT-spermatozoa with intact membranes × 100 divided by the % of pre-freeze spermatozoa with intact membranes) estimated. Significant differences were found among boars for freezeability (MI%) and motility post-thaw (%). Interestingly, significant correlations were found between the percentage of YO-PRO-1-positive spermatozoa and freezeability ( R = 0.440, p < 0.01), indicating this new triple staining can be used to safely disclose among ejaculates prior to freezing.

The Cryoprotective Effect on Frozen-thawed Boar Semen of Egg Yolk Low Density Lipoproteins

In order to protect the spermatozoa against cold shock, hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders for domestic animals. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). The effects of LDL on sperm quality of bull and northern pike (Esox lucius) after freezing-thawing have been reported, but no study has been made to evaluate the effect of LDL on boar sperm motility and other characteristics. The experiment was carried out to investigate the effect of LDL on the freezing of boar sperm in 0.25 ml straws. The aim was to evaluate the quality of boar spermatozoa cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Tris-citric acid-glucose (TCG) and Tris-citric acid-fructose (TCF), two basic freezing extenders containing egg yolk. Similarly, acrosome and plasma membrane integrity were also evaluated and compared to semen cryopreserved with TCG and TCF. Analysis of sperm quality after freeze-thaw showed that the motility, acrosome and plasma membrane integrity were improved with LDL in the extender, as compared to the TCG and TCF. The highest post-thaw integrity of acrosome and plasma membrane and motility were obtained with 9% LDL (w/v). Consequently, the optimum LDL concentration in the extender was 9%. It is also suggested that the concentration of LDL addition is important for the effect on boar sperm protection during freezing and thawing. The percentage of motile spermatozoa was significantly higher after freezing in 9% LDL than in TCG and TCF 54.4% versus 30.4% and 30.1% (p<0.05), respectively. The integrity of acrosome and plasma membrane were also significantly higher at 70.3% and 50.5% respectively with semen frozen in 9% LDL extender compared to TCG at 37.8% and 30.3% and TCF at 36.4% and 29.9%, respectively (p<0.05),. In conclusion, we propose that extender containing LDL extracted from hen egg yolk could be used as a cryoprotective media with a better efficiency than TCG and TCF. LDL improved boar semen quality, allowing better spermatozoa motility, acrosome and plasma membrane integrity after the freeze-thaw process. Furthermore, we found out that the extender with 9% LDL concentration significantly enhanced motility, acrosome and plasma membrane integrity of boar sperm after freezing and thawing. (Asian-Aust. J. Anim. Sci. 2006.

275 PENTOXIFYLLINE ADDED TO FREEZING EXTENDER HAS A DELETERIOUS EFFECT ON POST-THAW BOAR SPERM QUALITY AND IN VITRO FERTILIZATION RATE

Pentoxifylline, a methylxanthine derivative, is considered to be a hyperactivation and acrosome reaction-improving agent. The purpose of this study was to test how the addition of pentoxifylline to freezing extender influences post-thaw sperm motility and membrane integrity. The ability of thawed spermatozoa to fertilize in vitro-matured oocytes was also assessed. Pooled ejaculate sperm-rich fractions collected from three fertile boars were frozen in 0.5-mL straws after being diluted in lactose/egg yolk/glycerol/Orvus-ES-Paste extender (0 pentoxifylline = control) or the same extender supplemented with 2, 4, 8, 16, and 32 mM pentoxifylline. To evaluate post-thaw sperm survival, the percentage of total motile spermatozoa and rapid progressive spermatozoa (CASA system) and plasma membrane and acrosome integrity (flow cytometry) were assessed. Data from six replicates were analyzed in a split plot design using a PROMIXED model. The addition of 4, 8, 16, and 32 mM pentoxifylline to freezing extender significantly decreased progressive and total motility (P &lt; 0.001) compared with control (4.5/26.6%, 4.5/24.5%, 2.8/20.5%, 0.6/11.4%, and 13.2/49.7% for the 4, 8, 16, and 32 mM pentoxifylline groups and the control group, respectively). The same was observed for sperm viability; the percentage of viable spermatozoa with intact acrosomes was significantly lower (P &lt; 0.001) in pentoxifylline-treated groups compared with the control group, chiefly in the 16 mm, and 32 mM pentoxifylline groups (54, 11.6, and 6.2% for control, 16 and 32 mM, respectively). To evaluate in vitro fertilization parameters, cumulus-oocyte complexes were matured in BSA-free NCSU23 medium with 10% porcine follicular fluid, 0.1% cysteine, 10 ng EGF, 10 IU/mL eCG, and 10 IU/mL hCG, in a incubator at 39�C and 5% CO2. After 40-44 h of maturation, oocytes (n = 1067, in three replicates) were denuded of cumulus cells, washed, transferred to droplets (30 oocytes in 50 �L) of TBM medium supplemented with 2 mM caffeine and 0.2% BSA, and inseminated (2000 thawed sperm/oocyte). After a co-incubation period of 6 h, oocytes were washed and transferred to droplets (500 �L) of NCSU23 with 0.4% BSA for another 10-14 h, then fixed and stained for 72 h, and examined under a phase-contrast microscope. Data were analyzed as split plot design using a PROMIXED model. The addition of pentoxifylline to the freezing extender reduced significantly (P &lt; 0.001) the penetration rate (51.4, 17.5, 15.8, 17.8, 9.5, and 4.8% for the 0, 2, 4, 8, 16, and 32 mM pentoxifylline groups, respectively) and the efficiency (monospermic oocytes/total inseminated oocytes) of fertilization (34.8, 14.6, 14.7, 15.5, 7.9, and 4.8% for the 0, 2, 4, 8, 16, and 32 mM pentoxifylline groups, respectively) as compared with the control group (the first value in each of these two cases). It is therefore concluded that pentoxifylline added to the freezing extender has a deleterious effect on post-thaw boar sperm quality and on their ability to fertilize the oocytes in vitro. This work was supported by CICYT (AGL05-0471).

Effects of in vitro storage time and semen-extender on membrane quality of boar sperm assessed by flow cytometry

The Norwegian AI company Norsvin has used the short-term semen-extender BTS to extend and store boar semen since the late 1980s. Fertility results have been consistent when extended semen has been used for AI within 3 days after collection, however, from a production and economic point of view it is preferable that semen stored for up to 5 days can be used. The aim of this study was to compare membrane quality of sperm stored in BTS for 3 days with sperm stored in the long-term semen-extenders Androstar, Mulberry III and X-cell™ for 5 days. Using a split-sample design, plasma membrane- and acrosome-integrity were assessed flow cytometrically by use of Yo-Pro-1 and PNA-FITC, and fluidity and phospholipid asymmetry of the membrane were assessed by use of MC540 and Annexin V-FITC. Due to observed sperm fragmentation in Androstar after Day 1, the data for Androstar were excluded from the analyses. After 5 days of storage, the membrane quality of X-cell™-stored sperm was not statistically different from that of sperm stored in BTS for 3 days, while membrane quality of sperm stored in Mulberry III was statistically better on Day 5 compared to BTS on Day 3. In conclusion, Mulberry III and X-cell™ preserve sperm quality, as well as that of BTS on Day 3, for up to 5 days after collection.