Sperm-rich Fraction Research Articles

Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation?

Previous studies have shown sperm quality post-cryopreservation differs depending on the fraction of the seminal plasma boar spermatozoa are fortuitously contained in. As such, spermatozoa contained in the first 10 mL of the sperm-rich fraction (portion I) have better sustained handling procedures (extension, handling and freezing/thawing) than those contained in the ulterior part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction, portion II). However, those studies were performed using pooled samples. In the present study, individual ejaculates were used. Split ejaculates (portions I and II) from five boars were frozen and thawed using a conventional freezing protocol, followed by computer-assisted motility and morphology analysis (CASA and ASMA, respectively), as well as an Annexin-V assay for spermatozoa from each boar and ejaculate portion. Significant differences between portions were observed in all ASMA-derived variables, except in one boar. Also significant differences were observed between boars and ejaculate portions in sperm quality post-thaw. We identified, however, boars showing best results of motility and sperm membrane integrity post-thaw in portion I, while in other boar the best results was observed in portion II. It is concluded that the identification of the ejaculate portion more suitable to sustain cryopreservation in each individual boar may be a readily applicable and easy technique to diminish variation in sperm freezability among boars.

Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase.

In the present study the potential benefit of reactive oxygen species (ROS)-scavenging enzymes superoxide dismutase (SOD) and catalase (CAT) when cryopreserving boar spermatozoa was evaluated. Pooled ejaculate sperm-rich fractions collected from 3 fertile boars were frozen in a split design, after being extended in a conventional freezing extender (control) or the same extender supplemented with SOD (150 or 300 IU/mL, experiment 1), CAT (200 or 400 IU/mL, experiment 2), or SOD + CAT in combination (150 + 200 or 300 + 400 IU/mL, experiment 3). Irrespective of the concentration used, SOD and CAT, alone or in combination, significantly improved postthaw sperm survival, in terms of total sperm motility (assessed with CASA) and viability (assessed with a triple stain; propidium iodide/R123/fluorescein isothiocyanate-labeled peanut agglutinin). Moreover, CAT alone, at a concentration of 400 IU/mL, or in combination with SOD, at concentrations of 200 and 400 UI/mL, improved the ability of frozen-thawed spermatozoa to produce embryos in vitro (zygote cleavage and blastocyst formation as end points). Additional data of ROS generation (luminol- and lucigenin-dependent chemiluminescence) and membrane lipid peroxidation (malondialdehyde [MDA] production) indicated that SOD and CAT reduced postthaw ROS generation by boar spermatozoa, without any influence on MDA production.

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

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.

92 EVALUATION OF BOAR SPERM FUNCTIONALITY AFTER A CUSHIONED CENTRIFUGATION TECHNIQUE

Separation of sperm from seminal plasma is required in most semen freezing procedures. Semen is typically subjected to centrifugation to concentrate sperm into a pellet and allow removal of the seminal plasma prior to dilution in freezing extender. Centrifugation is a relatively effective method to recover sperm, however, the process also causes considerable sperm damage. The use of a dense, inert, and isotonic solution as a cushion in the bottom of the centrifuge tube allows a greater centrifugation speed to be applied and results in greater sperm recovery. The aim of the present work was to evaluate the effects of this cushioned centrifugation technique on in vitro sperm viability and functionality. Sperm-rich fractions from 16 fertile boars were diluted and cooled to 15�C; then subsamples were centrifuged by one of two different techniques. A standard method (SM), 800 g for 10 min in 50-mL tubes (Westendorf et al. 1975 Dtsch. Tier�rztl. Wschr. 82, 261-267) and a cushioned method (CM), 1000 g for 20 min using 45 mL of diluted semen on 5 mL of an isotonic iodixanol solution (60% w/v gradient) were performed. Sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378-391) to detect changes in lipid packing disorder of the plasma membrane. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of reactive oxygen species (ROS). A final set of sperm samples was stained with peanut aggultinin-fluorscein isothiocyanate (PNA-FITC) and propidium iodide to evaluate the acrosome reaction. All of these parameters were evaluated by flow cytometry before and after centrifugation. ANOVA analysis revealed that centrifugation altered lipid packing disorder and viability. Raw semen (RS) had a larger number of viable low lipid disorder sperm than centrifuged semen (RS = 86.9a vs. SM = 81.64b vs. CM = 80.6b, P < 0.01) and a decreased number of dead sperm cells (RS = 9.5a vs. SM = 15.0b vs. CM = 16.3b, P < 0.01). However, the cushioned and standard centrifugation methods yielded similar results for all the parameters measured. No significant differences were found for generation of ROS or in the number of sperm exhibiting the acrosome reaction. In conclusion, compared to the standard centrifugation method, this simple cushioned modification is a more efficient means of processing boar semen for freezing because significantly less sample losses are detected; also, it provides similar levels of sperm viability and functionality, and consequently a higher number of doses per ejaculation can be produced.

135 THE EFFECT OF DIFFERENT TREATMENTS OF PORCINE EJACULATED AND EPIDIDYMAL SPERMATOZOA ON ROS GENERATION

The production of pig embryos in vitro is still relatively inefficient compared with results obtained with oocytes matured and fertilized in vivo. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of embryos produced by IVM-IVF (Kikuchi et al. 2002 Biol. Reprod. 1033-1041). Between factors affecting polyspermy are the sperm procedures before IVF. Usually, these procedures including centrifugations that increase reactive oxygen species (ROS) formation in spermatozoa. ROS play an important role in sperm physiology including capacitation. Physiological concentrations of ROS have been proposed to enhance sperm capacitation by increasing cAMP synthesis and by inhibiting protein tyrosine phosphatases whilst activating tyrosine kinases. In general, epididymal spermatozoa appear to be able to capacitate and fertilize eggs in vitro much more easily than ejaculated spermatozoa (Yanagimachi Mammalian Fertilization. In: The Physiology of Reproduction, Raven Press 1988; 135-182). In this study, we investigated how different sources (ejaculated spermatozoa vs. epididymal spermatozoa) and sperm capacitating methods, usually employed in porcine IVF, could be affect ROS generation. Sperm-rich fractions from five fertile boars and sperm from five different epididyimides were used. The semen samples were then: (i) washed in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 0.1% BSA, (ii) left unwashed, or (iii) washed on a Percoll (Pharmacia, Uppsala, Sweden) gradient (Mat�s et al. 2003 Reproduction 125, 133-141). Production of ROS was measured by incubating the spermatozoa in the in vitro fertilization medium (TALP) in the presence of 0.7 �m 22,72-dichlorodihydrofluorescein diacetate at 37�C under 5% CO2 in 100% humidified air. Every 15 min (from 15 to 135) the samples were analyzed and evaluated by flow cytometry. Measurements were expressed as the mean green intensity fluorescence units and it was used as index of ROS generation (Gadea et al. 2005 J. Androl. 26, 396-404). ANOVA analysis revealed a significant effect of sperm treatment on the ROS generation (P < 0.001). The highest value was obtained in sperm washed on a Percoll gradient and the lowest in unwashed semen. When ejaculated vs. epididymal semen was analyzed, the same tendency was observed in both. However, the values were always lower in epididymal semen than in ejaculated semen (P < 0.001). As a conclusion, ROS generation is different between treatments and between semen procedures for the time interval studied, and this finding may help to explain the different outcome in IVF among laboratories. This work was supported by Ministerio de Educaci�n y Ciencia, AGL2003-03144.

Evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapours) freezing

Three ejaculates were collected from each of five dogs. After initial evaluation, the sperm-rich fractions were diluted to 100 x 10(6) spermatozoa x mL(-1) in two steps with an egg yolk-TRIS extender containing a final concentration of 5% glycerol and 0.5% Equex STM paste. Half of the 0.5 mL straws obtained from each ejaculate were frozen on nitrogen vapours (4 cm above the liquid surface) ("rapid freezing"), while the other half was frozen in a biological freezer at a rate of 0.5 degrees C x min(-1) between 5 degrees C and -10 degrees C and of 8 degrees C x min(-1) between -10 degrees C and -60 degrees C, followed by immersion in liquid nitrogen ("slow freezing"). After an average storage of 30 days, the straws were thawed in a water-bath at 37 degrees C for 1 min. Progressive motility was subjectively estimated hourly for 8 h on semen incubated at 38 degrees C. Immediately after thawing and after 2 h of incubation, motility parameters were also measured by a motility analyser. Sperm membrane function and chromatin stability were assessed immediately post-thaw, using the hypo-osmotic swelling test and acridine orange staining, respectively. Slow freezing significantly improved total post-thaw motility, which showed a slower decline over time, although spermatozoal average path and straight line velocity were lower compared to the fast rate. Also the number of intact membrane spermatozoa was significantly higher in slow-frozen samples while the proportion of spermatozoa with single-stranded DNA was minimal after both freezing procedures.

89 EVALUATION OF A CUSHIONED CENTRIFUGATION TECHNIQUE FOR PROCESSING BOAR SEMEN FOR FREEZING

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.

266 ANTIOXIDANT CAPACITY OF BOAR SEMINAL PLASMA

It has been established that antioxidants in seminal plasma play an important role in protecting the spermatozoa against oxidative stress-induced damage. This study was conducted to measure the total antioxidant capacity (TAC) of boar seminal plasma. Fifty-four ejaculates were collected from 17 mature boars of proven fertility by the gloved-hand technique. Ejaculates were collected separately in different fractions (pre-sperm, sperm-rich, and post-sperm) according to their macroscopic (color) characteristics. After centrifugation (2400g for 3 min), the sperm pellet was discarded; the supernatant was recentrifuged and filtered through a 10-μm nylon mesh filter to remove debris or clumped spermatozoa. The seminal plasma was frozen at -20°C until further use. After thawing at room temperature, seminal plasma aliquots of 5 μL were immediately assessed for total antioxidant capacity. TAC was measured using the ABTS/H2O2/HRP decoloration method (Cano A et al. 2000 Redox Report 5, 365–370) which allows differentiation between hydrophilic and lipophilic antioxidant activity capacity. TAC units were expressed as micromolar (μM) Trolox equivalents. Data were analyzed using ANOVA. Only the hydrophilic activity was measurable, with the lipophilic activity being undetected. The overall TAC of seminal samples (mean ± SEM) was 1623.7 ± 56.28 μM, ranging from 674 to 2428 μM. Different TACs were observed among males (P < 0.05) and between ejaculates of the same male (P < 0.05). Ejaculate fraction had a significant effect (P < 0.001) on the TAC levels. The post-sperm fraction had a significantly lower TAC level (1104.09 ± 57.66 μM) than the pre-sperm and sperm-rich fractions (1611.95 ± 153.68 μM and 1356.136 ± 72.47 μM, respectively, P < 0.001). In conclusion, hydrophilic antioxidant activity represented the main contribution to the TAC in boar seminal plasma, showing differences among males, between ejaculates of the same male, and also between the different ejaculate fractions. This work was supported by CICYT (AGF98-0533; AGL01-0471) and INIA (RZ01-019).

142 INTRACELLULAR CALCIUM UPTAKE AS A RESPONSE TO DIFFERENT CAPACITATION TREATMENTS IN EJACULATE AND EPIDIDYMAL BOAR SPERMATOZOA

Both cauda epididymal and ejaculated spermatozoa are considered fully mature, although these two types of spermatozoa do not necessarily show the same behavior in vitro. It has been reported that both types of spermatozoa fertilize eggs in vitro at the same rate, but, in general, epididymal ones achieve this objective easier than the ejaculated ones. Intracellular Ca2+ influx is one of the crucial biochemical events occuring capacitation and Ca2+ requirements for capacitation varies among different species. In this study, we investigated how different source of spermatozoa (ejaculated vs. epididymal) and commonly employed sperm capacitating methods can affect calcium uptake. Sperm-rich fractions from seven fertile boars and sperm from seven different epididymides were used. Semen samples were kept unwashed (method A), washed in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 0.1% BSA (method B), or washed on a Percoll gradient (method C) (Mat�s et al. 2003 Reproduction 125, 133-141). To measure intracellular free Ca2+, spermatozoa, treated as described above, were incubated with 2.5 �m fura-1/AM in a non capaciting medium (Tardif et al. 2003 Biol. Reprod. 68, 207-213) for 45 min at 37�C. Then, spermatozoa were resuspended in TALP medium, incubated (5% CO2, 38.5�C) for a further 60 min and then analyzed in a fluorescence spectrofluorometer with excitation wavelength set at 340-880 nm and emission held at 510 nm. The calculation of intracellular free Ca2+ was performed according to the equation of Grynkiewicz et al. (1985 J. Biol. Chem. 260, 3440-3450). Results showed that calcium uptake is affected by treatment and semen source (P < 0.001). The intracellular free Ca2+ concentrations (nM) in ejaculated semen and in epididymal spermatozoa were 269.52 vs. 208.52, 1134.58 vs. 137.37 and 1224.79 vs. 216.54 for A, B, and C methods, respectively. As a conclusion, it can be stated that sperm capacitation treatment affects calcium uptake. In addition, capacitation pathways may be modified or regulated in some way by seminal plasma, thus increasing intracellular calcium levels in ejaculated sperm (methods B and C) in comparison to those in epididymal spermatozoa. This work was supported by Ministerio de Educaci�n y Ciencia, AGL2003-03144.

97 DIFFERENCES IN SPERM CHROMATIN STRUCTURE AMONG GOOD AND BAD BOAR SPERM FREEZERS

Inter- and intra-boar differences in sperm freezability are observed independent of the sperm quality before freezing, the breed, or the genetic line. This study aimed to determine whether boars with different post-thaw sperm quality also show differences in sperm DNA integrity. Sperm-rich fractions (3 to 10 ejaculates per boar) from 19 fertile mature boars were extended in Beltsville thawing solution (BTS) and cooled to 17�C for 16 h. Then, samples were centrifuged at 2400g for 3 min, extended in freezing extender (lactose/egg yolk/glycerol/Equex STM; Nova Chemical Sales, Inc., Scituate, MA, USA) to a final concentration of 1 � 109 spermatozoa/mL, dispensed into 0.5 mL straws, and frozen in a programmable cell freezer at a rate of -20�C min. Thawing was carried out in a water bath at 37�C for 20 s. Frozen-thawed spermatozoa were evaluated for progressive sperm motility (PSM) using a computer-assisted sperm analysis (CASA) system, and sperm viability (PMI) using flow cytometry. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all boars into two groups, categorized as good (n = 10; >60% PSM and PMI) or bad (n = 9; <40% PSM and PMI) based on their sperm freezability. Post-thaw sperm quality was consistent for different ejaculates within boars (P < 0.05). The DNA-integrity of frozen-thawed spermatozoa was evaluated according to the sperm chromatin structure assay (SCSA; Evenson et al. 1980 Science 210, 1131-1133). All SCSA variables (X mean, DNA fragmentation index (DFI), and the standard deviation of the DFI), were significantly higher for bad freezers (P < 0.001). The percentage of spermatozoa with abnormal chromatin structure ranged from 1.06 to 3.42% for good and 3.06 to 6.04% for bad freezers. Although these differences exist between good and bad sperm freezers, and can only to some extent be the product of cryopreservation, the levels of affected spermatozoa can not explain the differences on post-thaw sperm survival seen in the two categories of sires. This work was supported by CICYT, AGL05-0471 (Spain), SLF and Formas (Sweden).

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 < 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 < 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 < 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).

11 TIME OF INSEMINATION RELATIVE TO OVULATION EXPLAINS FERTILITY VARIATIONS OFFROZEN - THAWED SPERMATOZOA BETWEEN FARMS

Swine fertility after AI with frozen-thawed spermatozoa varies between trials. As thawed spermatozoa have an extremely limited life span in the female genital tract, fertility of frozen-thawed spermatozoa depends mainly on the time of insemination relative to ovulation. The objective of this study was to evaluate whether the time of insemination relative to ovulation could explain the farm differences in fertility when frozen-thawed spermatozoa are used. Pooled sperm-rich fractions collected from three mature Pietrain boars were diluted in lactose/egg-yolk/glicerol/Orvus-ES-Paste extender, loaded in 0.5-mL straws (1 × 109 cells/mL), and frozen under controlled conditions (Carvajal et al. 2003 J. Androl. 25, 389–396). Thawing was conducted in a waterbath at 37°C for 20 s. Inseminations were performed using the deep intrauterine insemination technique (Martínez et al. 2002 Reproduction 123, 167–170) with 1 × 109 thawed spermatozoa (post-thaw motility >50%) diluted in 5 mL of Beltsville thaw solution (BTS). Ninety-seven and 82 weaned sows (parity 2–7) in farms A and B, respectively, were twice inseminated at 30 and 36 h after onset of estrus (estrus detection was performed twice a day by allowing females nose-to-nose contact with a mature boar and by applying back pressure). At insemination time, both ovaries were checked for ovulation by transrectal ultrasonography and sows were classified into three groups: F sows (follicles visible during the two examinations), O sows (ovulation visible during at least one examination), and C sows (corpora lutea visible during both examinations). Data were analysed with ANOVA and chi-square test, and are reported as % or mean ± SEM. Overall farrowing rates differed (P < 0.01) between farms: 70.1% (68/97) and 51.22% (42/82) in farms A and B, respectively. Litter size did not differ (P > 0.05) between farms (9.18 ± 0.24 and 9 ± 0.39 in farms A and B, respectively). Distribution of sows among F, O, and C groups differed (P < 0.05) between farms. Seventeen (17.52%), 70 (72.16%), and 10 (10.31%) sows in farm A and 33 (40.24%), 24 (29.27%), and 25 (30.49%) sows in farm B were classified as F, O, and C, respectively. Fertility in F, O, or C sows did not differ (P > 0.05) between farms. Farrowing rates and litter size in O sows (82.98% and 9.45 ± 0.23) were higher (P < 0.05) than in F (48% and 8.67 ± 0.54) and C (48.57% and 7.55 ± 0.62) sows. We can conclude that time of insemination relative to ovulation explains fertility differences between farms when frozen-thawed spermatozoa are used. This work was supported by INIA (RZ01-019) and INFO (CARM).

Motility and fertility of alginate encapsulated boar spermatozoa

Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 °C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16–18 °C, and then were stored at 5 °C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 °C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24 h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 °C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8 h incubation at 37 °C after storing for over three days ( P < 0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability.

Boar spermatozoa in the oviduct

In the pig, a functional tubal sperm reservoir (SR) is established before ovulation to ensure availability of suitable numbers of viable spermatozoa for fertilization. The boar's large ejaculate is split: most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF fraction containing increasing amounts of the spermadhesin PSP-I/PSP-II-rich seminal vesicle secretion. This heterodimer acts as leukocyte chemoattractant both in vitro and in vivo, contributing to the phagocytosis of those spermatozoa not reaching the SR. Sequential ejaculate deposition of marked spermatozoa and SR screening showed that most spermatozoa in the SR arose from the fortuitous PSP-poor, first portion of the SRF fraction, escaping phagocytosis and replenishing the SR within 2–3 h. The SR-sperm numbers diminish gradually in relation to ovulation, spermatozoa being continuously redistributed toward the upper isthmus. In vitro, only uncapacitated spermatozoa bind to epithelial explants, suggesting that the SR influences sperm capacitation. In vivo, most viable spermatozoa – usually harbored in the deep furrows in the pre- or peri-ovulatory SR during spontaneous standing estrus – are uncapacitated, but capacitation significantly increases after ovulation. Pre-/peri-ovulatory SR spermatozoa promptly capacitate in vitro when exposed to the effector bicarbonate, an influence that can be reversed by co-incubation with SR fluid or its component hyaluronan. Fluid collected from the ampullar segment (rich in bicarbonate) induces capacitation in vitro. In conclusion, the lack of massive sperm capacitation in the SR and the diverse individual response to capacitation shown by tubal spermatozoa would relate both to the insurance of full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation in the upper oviduct, thus maximizing the chances of normal fertilization.

Characterization and localization of membrane vesicles in ejaculate fractions from the ram, boar and stallion.

Membrane vesicles, separated by differential centrifugation from the seminal plasma, were detected in the sperm-rich ejaculate fractions of four boars and three stallions, and in the whole ejaculates of seven rams. The volume and percentage of vesicles, determined by a stereological technique, were higher in the sperm-rich than in the post-sperm-rich fractions of the boar and stallion ejaculates, and no vesicles were detected in the pre sperm-rich fractions. Vesicles were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For boar, stallion and ram semen, the mean (+/- s.e.m.) vesicle diameters were 130.9 +/- 3.22 (range 18-577), 164.1 +/- 4.42 (range 15-671) and 159.7 +/- 2.92 nm (range 22-986), respectively, although they were not significantly different (p = 0.709). The vesicles had approximately round (TEM) or spherical shape (SEM), were surrounded by single, double or multi-laminar membranes, and were trapped within ample amorphous material, sometimes containing short, flattened membranous elements. The majority of the vesicles had a clear interior but some contained granule-dense material. Ram membrane vesicles, purified from ultracentrifuged plasma by size exclusion chromatography, kept their round shape and the amorphous material was less evident compared with the sections taken before purification. This is the first report to identify seminal plasma membrane vesicles in the different fractions of ejaculated semen in the boar and stallion, and confirms their presence in ram seminal plasma. The origin and function of these vesicles are yet to be elucidated.

Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture.

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.

289EFFECT OF FERTILIZATION TIME OF PIG OOCYTES MATURED IN-VITRO BY BOAR SPERM STORED AT 4°C

The use of boar sperm stored at 4°C may be a useful tool for in vitro production of pig embryos. Therefore, this study was undertaken to investigate the effect of fertilization time of pig oocytes matured in-vitro by boar sperm. The sperm-rich fraction (30–60mL) was slowly cooled to room temperature (20–23°C) by 2h after collection. Semen was transferred into 15-mL tubes, centrifuged at room temperature for 10min at 800g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5mL of the LEN (11.0g lactose hydrate, 20mL egg yolk, 0.05g N-acetyl-D-glucosamine and 100mL distilled water) diluent to provide 1.0×109 spermmL−1 at room temperature. The resuspended semen was cooled in a refrigerator to 4°C. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Oocytes were inseminated with boar sperm stored at 4°C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9h in 500μL TBM fertilization media with 1×106mL−1 sperm concentration. Thereafter, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture of 6, 48 and 144h, fixed and stained for the evaluation of fertilization parameters and developmental ability. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times of 6 and 9h than in those of 1 and 3h. The percentage of polyspermic oocytes was highest in fertilization time of 9h compared with other incubation times. The rates of cleaved oocytes were higher in the fertilization times of 6 and 9h (85.0 and 84.6%) compared with those of 1 and 3h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time of 6h (33.6%) than in that of 1, 3 and 9h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9±3.3, 27.6±2.7, 26.3±2.2 and 24.4±1.8 in the fertilization times of 6, 9, 3 and 1h, respectively. In conclusion, we found out that boar sperm stored at 4°C could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend the coincubation time of 6h in 500μL TBM fertilization medium with 1×106mL−1 sperm concentration for in vitro fertilization of pig oocytes matured in vitro.

92THE ADDITION OF SEMINAL PLASMA FROM INDIVIDUAL BOARS TO FREEZING EXTENDER CAN IMPROVE THE POST-THAW SPERM SURVIVAL

Contradictory results have been reported about the effect of seminal plasma (SP) on the freezability of mammalian spermatozoa. In pigs, current methods for sperm cryopreservation involve removing seminal plasma. Therefore, no conclusive evidence of the potential effect of SP on the freezability of boar spermatozoa has been reported. In this study, we evaluate the effect of the addition of low concentrations of SP from individual boars to the freezing extender on post-thaw sperm survival. Sperm cryopreservation procedure included: dilution of sperm-rich fraction in Beltsville Thaw Solution extender (BTS), cooling to 17°C for 16h, centrifugation at 2400g for 3min, dilution in lactose/egg-yolk/glycerol/Equex Stem (freezing extender) to a final concentration of 1×109 spermmL−1, dispensing into 0.5-mL straws, and freezing in a programmable cell freezer at 20°Cmin−1. Thawing was carried out in a waterbath at 37°C for 20s. Post-thaw sperm survival was assessed by progressive sperm motility (PSM) using a CASA system (SCA); plasma membrane integrity (PMI) and acrosome membrane integrity (AMI) were assessed by flow cytometric procedures (SYBR-14/PI and FITC-PNA/PI, respectively) at 30 and 150min post-thawing in BTS-diluted thaw spermatozoa held in a waterbath at 37°C. Four individual seminal plasma donors (SP1 to SP4) were selected in a preliminary study in which 48 ejaculates from 12 boars (4 ejaculates/boar) were cryopreserved. Then the boars were classified into 3 groups (good, moderate and bad freezers) based on their post-thaw sperm survival. SP1 and SP2 were good freezers (&amp;gt;60% PSM and PMI), SP3 was a moderate freezer (40–60% PSM and PMI) and SP4 was a bad freezer (&amp;lt;40% PSM and PMI). In the main experiment, pooled sperm-rich fractions collected from 9 mature hybrid boars were divided into five aliquots and each was diluted with freezing extender supplemented with 0% (control) or 10% of SP (1–4). Data from eight replicates were analyzed as a split plot design using a PROMIXED model. The addition of SP to freezing extender had a significant effect (P&amp;lt;0.05) on post-thaw sperm survival compared to control. Moreover, there were significant differences (P&amp;lt;0.05) between SP donors. PSM, PMI and AMI were significantly (P&amp;lt;0.05) higher in SP1 (56.71±4.30; 57.16±4.01 and 57.22±4.01, respectively) and SP2 (59.48±4.30; 60.17±4.01 and 60.05±4.01, respectively) compared to control (50.39±4.30; 49.98±4.01 and 49.54±4.01, respectively). There were no differences (P&amp;gt;0.05) between SP3, SP4 and control. These results indicate that the addition of SP from particular boars (good freezers) to freezing extender may improve post-thaw sperm survival. Individual differences in the SP composition should explain the above results. Supported by INIA (RZ01-019) and MCYT (AGL2001-0471).

Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen–thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates ( P<0.05). In Experiment 2, semen from four stallions (60–70 ejaculates per stallion) was collected either once daily or two times 1 h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24 h at 5 °C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1 h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (≥12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased ( P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 °C, percentage of membrane-intact sperm in fresh semen ( P<0.05) as well as motility and percentage of membrane-intact sperm of frozen–thawed sperm ( P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom ( P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (≥1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5–1 or <0.5. FR of mares inseminated with stallions having 0.5–1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1–1.5 days or >2.5 days.

MEMBRANE STATUS AND IN VITRO CAPACITATION OF PORCINE SPERM PRESERVED IN LONG-TERM EXTENDER AT 16°C

Preservation of porcine semen in long-term extenders at 15-18°C for more than 5 days results in decreased farrowing rates and reduced litter size after artificial insemination, despite the high progressive motility rates of sperm. To improve this preservation system it is necessary to understand sperm physiology under storage conditions. The purpose of this study was to determine the effect of storing diluted porcine semen (during 0, 2, 4, 6, and 8 days) on the sperm membranes status and the ability of sperm to respond to in vitro capacitation treatment. Ten semen samples from 5 adult boars were analyzed. Two aliquots were obtained from the sperm-rich fraction: one was used to assess fresh semen and the other was diluted in Reading extender and stored at 16°C. Both semen samples were stained with chlortetracycline to assess the status of sperm membranes and with Hoechst 33258 to determine viability. Semen storage for 4-8 days increased the proportion of prematurely capacitated sperm. After 4 days of storage, in vitro capacitation treatment did not increase the percentage of capacitated sperm, but increased the percentage of acrosome reacted sperm. This phenomenon could explain the reduced fertilizing ability of porcine semen stored at 16°C for over 4 days, in spite of the acceptable sperm viability and progressive motility.