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Theriogenology 71 (2009) 491–498 www.theriojournal.com
Seminal plasma damages sperm during cryopreservation, but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality T. Okazaki a,b, S. Abe a, S. Yoshida a, M. Shimada b,* a
Smaller Livestock and Environment Section, Livestock Research Institute, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Bungo-ono, Oita 879-7111, Japan b Department of Applied Animal Science, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama Higashi-Hiroshima, Hiroshima 739-8528, Japan Received 21 May 2008; received in revised form 18 August 2008; accepted 25 August 2008
Abstract To determine the effects of seminal plasma during and after cyopreservation on post-thaw sperm functions in semen from poor freezability boars, seminal plasma was removed immediately after collection, and sperm was subjected to cooling and freezing. Removal of seminal plasma did not significantly affect post-thaw sperm motility in good freezability boars; however, in boars with poor freezability, it increased post-thaw motility relative to control sperm cooled with seminal plasma (64.5 3.4% vs. 30.9 3.1%, P < 0.01). Freezing sperm without seminal plasma increased both loss of the acrosome cap (37.5 1.6% vs. 18.4 2.8%, P < 0.01) and expression of a 15 kDa tyrosine-phosphorylated protein (capacitation marker) in thawed sperm relative to controls; the addition of 10% (v/v) seminal plasma to the thawing solution significantly suppressed both changes and increased conception rate to AI (70% vs. 9% in the control group, P < 0.05). In conclusion, our novel cryopreservation and thawing method increased the success of AI with frozen-thawed porcine semen, particularly from boars with poor post-thaw semen quality. # 2009 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Seminal plasma; Artificial insemination; Pig; Spermatozoa
1. Introduction Cryopreservation of boar spermatozoa offers an effective means of long-term storage of important genetic material. Recently, we and other groups reported a high conception rate (70–80%) by AI using boar spermatozoa cryopreserved using a modification of previously described cryopreservation methods  and * Corresponding author at: Laboratory of Animal Reproduction, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan. Tel.: +81 824 24 7899; fax: +81 824 24 7988. E-mail address: [email protected]
(M. Shimada). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.08.014
through the development of a novel sperm infusion method . However, it is well known that the quality of frozen-thawed spermatozoa and the conception success rate are also dependent on unique characteristics of individual boars [3–6]. Thurston et al.  categorized sperm head morphology by Fourier descriptors, and detected a significant association between the head morphology of fresh spermatozoa and the motility of sperm after freezing and thawing. Additionally, they identified molecular markers linked to genes controlling semen freezability by amplified restriction fragment length polymorphism technology , suggesting that the freezability of boar sperm was affected by genetic factors.
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In a study in which semen was collected as two portions, the sperm-rich fraction and the post sperm rich fraction, in several boars there were significant differences between fractions in post-thaw sperm motility . In addition, the freezability of epididymal sperm is higher than that of ejaculated sperm in boars . Although the poor freezability of boar sperm is potentially affected by genetic background, we speculated that the effects of seminal plasma on sperm after ejaculation may be another factor. Removing seminal plasma immediately after semen collection is one of the available techniques to improve motility of frozen-thawed sperm and in vitro fertility competence in miniature pigs . However, frozenthawed epididymal porcine sperm does not have high in vivo fertilization competence [Okazaki and Shimada, unpublished data, 12]. Additionally, it has been reported that seminal plasma is required to protect sperm against a spontaneous capacitation-like reaction during the thawing process . Thus, we hypothesized that the presence of seminal plasma during freezing is not a critical determinant of post-thaw sperm quality in boars with good freezability, but has deleterious effects in those with poor freezability, and that seminal plasma is essential to maintain fertility competence during thawing. Consequently, we designed the following novel freezing and thawing method to preserve sperm from boars exhibiting poor sperm freezability. First, seminal plasma was removed from the sperm by centrifugation immediately after semen collection. Second, the sperm was frozen using a conventional method , and the cryopreserved sperm was thawed in a medium containing seminal plasma collected from a boar with proven high reproductive performance. The thawed sperm was then used for AI. To evaluate this freezing and thawing method, we analyzed frozenthawed sperm motility, capacitation and acrosomal status in vitro and fertilization competence in vitro and in vivo. 2. Materials and methods 2.1. Media and seminal plasma Routine chemicals and reagents were obtained from Nakarai Chemical Co. (Osaka, Japan) and Sigma Chemical Co. (Sigma; St. Louis, MO, USA). The pre-treatment solution contained 0.33 M Dglucose, 12.8 mM trisodium citrate dihydrate, 14.3 mM sodium hydrogen carbonate, 9.9 mM EDTA-2Na, 1000 U/mL penicillin G potassium, and 1 mg/mL streptomycin sulfate.
Niwa and Sasaki freezing extender was used in this study, with some modifications (mNSF1, the osmolality was changed from 300 mOsm/kg to 400 mOsm/kg ). Orvus Es Paste (Miyazaki Chemical Sales, Ltd., Tokyo, Japan) at a concentration of 1.5% (v/v) and glycerol at a concentration of 4% (v/v) were added to mNSF1 (final glycerol concentration 2% (v/v)), which were then used for the second dilution (mNSF2) at 5 8C before freezing. Modena solution  was used as the thawing solution. The sperm incubation medium for the investigation of spontaneous capacitation and acrosomal status was Modena solution without EDTA-2Na (pH 7.0). To obtain seminal plasma for Experiment 3 (described below), spermatozoa recovered from highfertility boars (>80% conception rate with fresh semen) were removed from the semen within 15 min after collection by centrifugation at 700 g for 10 min. The supernatant was removed from the pelleted spermatozoa, centrifuged again at 1500 g for 30 min, and used as the seminal plasma added to the thawing solution. 2.2. Semen collection and freezing and thawing procedure Twenty mature Landrace and Large White boars, aged 15–28 mo, were used in this study. The sperm-rich fraction was collected weekly from each boar using the gloved-hand technique. The sperm-rich fraction was filtered thorough double gauze. The semen was directly diluted in pre-treatment solution (1:1, pre-treatment solution: semen) and held for 2 h at 15 8C. The semen was then centrifuged for 5 min at 800 g to remove the pre-treatment solution. The precipitated spermatozoa were gently resuspended with mNSF1, and cooled slowly from 15 to 5 8C over 1.5 h. Then the sperm suspension was diluted in the same volume of mNSF2 (1 109 sperm/mL) and transferred into a 0.5 mL plastic straw. The straws were placed in liquid nitrogen vapor for 10 min, and finally stored in liquid nitrogen. For thawing, the straw was transferred to water at 60 8C and kept there for 8 s, diluted in 4.5 mL of Modena solution, and then incubated at 37 8C for up to 6 h. The final concentration of the sperm was 1 108 spermatozoa/mL. 2.3. Evaluation of sperm motility, capacitation and acrosomal status After 1-, 3- and 6-h incubations of frozen-thawed spermatozoa in Modena solution at 37 8C, sperm
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motility was assessed using a computer-assisted sperm motility analysis (CASA) system (Version 8.1, Hamilton Thorne Biosciences, Beverly, MA, USA) . Three ejaculates from each boar were analyzed. Briefly, for each sample, a 5 mL aliquot of semen was placed on the analyzer’s Makler chamber, which was maintained at 37 8C during analysis. Three fields were selected for computer-assisted analysis. Total sperm motility (at least 200 motile spermatozoa were analyzed in every sample) was defined as the percentage of spermatozoa that had any form of motility. Protein samples from spermatozoa incubated with EDTA-2Na-free Modena solution (thawing method and dilution were performed as described above) were prepared by homogenization in Laemmli sample buffer . The samples were heated at 100 8C for 5 min, and 20 mL of each sperm extract was loaded in each lane (1 106 spermatozoa/lane) of a 10% SDS-polyacrylamide gel. Tyrosine-phosphorylated proteins were detected using an anti-phosphotyrosine antibody (PTyr-100, 9411, Cell Signaling, 1:5000) as described by Shimada et al. . Sperm capacitation was analyzed in triplicate using three different boars. The acrosomal status of spermatozoa was measured using FITC-labeled peanut agglutinin (FITC-PNA, Sigma) and PI staining . Frozen-thawed spermatozoa were diluted 1:10 (v/v) with Modena solution, after which the mixture was spread over the slides and airdried at room temperature. Samples were then fixed with absolute methanol for 10 min at room temperature, and 30 mL of FITC-PNA solution (100 mg/mL) in PBS was spread over each slide. The slides were then incubated in a dark, moist chamber for 20 min at 37 8C. Following a rinse with PBS and air-drying, slides were mounted using VectaShield with PI (Vector Laboratories Inc., Burlingame, CA, USA). More than 200 spermatozoa were evaluated for every sample. 2.4. In vitro fertilization In vitro fertilization was carried out according to Shimada et al. . Cumulus-oocyte complexes (COCs) were collected from 3 to 5 mm follicles and cultured for 44 h with porcine FSH (Sigma F-2293) and 1.0 IU hCG (Veterinary Puberogen, Sankyo, Tokyo, Japan). The COCs were then washed three times with fertilization medium [modified Tris-buffered medium (mTBM) supplemented with 0.1% (w/v) BSA (Fraction V; Sigma A 7888) and 1 mM caffeine (Sigma)]. Twenty COCs were placed in 50-mL drops of fertilization medium covered with mineral oil in a 35 mm 10 mm polystyrene culture dish. The dishes were kept in an
incubator for 30 min, until spermatozoa were added for fertilization. Frozen spermatozoa were thawed and washed by centrifugation at 700 g for 5 min in washing medium [mTBM supplemented with 0.1% (w/ v) BSA (Fraction V; Sigma A 7888)]. The sperm pellet was resuspended and pre-cultured for 90 min in preculture medium [mTBM supplemented with 10% (v/v) FCS and 1 mM caffeine] at a concentration of 2 108 spermatozoa/mL. The pre-cultured spermatozoa were diluted to 2 106 spermatozoa/mL with fertilization medium, and 50 mL of this suspension were added to the 50 mL of fertilization medium that contained oocytes (final concentration of sperm, 1 106 spermatozoa/mL). Oocytes were co-cultured with spermatozoa at 39 8C in an atmosphere of 5% CO2 in air for 6 h. Putative fertilized oocytes were washed three times with culture medium and incubated in 100-mL drops of the medium for 12 h. At the end of culture, oocytes were mounted on slides, fixed with acetic acid/ethanol (1:3) for 48 h and stained with aceto-lacmoid. Oocytes with single swollen or unswollen sperm heads in the ooplasm were considered to have been fertilized. The culture medium for the zygotes was NCSU37 containing 0.4% BSA (Sigma, Fraction V; A-8022). 2.5. Artificial insemination Synchronization of follicular development and ovulation was induced in Landrace sows by injection of 1000 IU of eCG (VETERINARY PEAMEX, 1000 U, Sankyo, Tokyo, Japan) 24 h after weaning. At 72 h post eCG, 500 IU of hCG were injected. Estrus detection was performed twice a day (09:00 and 16:00 h), beginning 2 days after eCG administration by allowing nose-to-nose contact with a mature boar and applying back pressure. Forty hours after hCG administration, sows were randomly distributed into test groups (see below), and the sows of each group were singly artificially inseminated using 50 mL of diluted frozen-thawed sperm. Ten straws of frozen sperm (total 5 mL) were thawed and then diluted in 45 mL of Modena solution (5 109 spermatozoa/50 mL). 2.6. Conception rate and number of implantation sites Confirmation of conception was determined by ultrasongraphy (Aloka, SUPER EYE, SSD-500, Tokyo, Japan) at Day 25 (AI = Day 0). We defined the conception rate as the number of pregnancies/total
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inseminated sows. Pregnant sows were slaughtered at a local abbatoir on day Day 30 of pregnancy, and the reproductive tract was recovered. The numbers of implantation sites, including live and dead foetuses, were counted.
data of conception rate were analyzed using the Chisquare test. P < 0.05 was set as the level of significance. Data are presented as mean S.E.M.
2.7. Experimental design
3.1. Experiment 1: the classification of boars as poor or good freezability
In Experiment 1, cryopreserved spermatozoa were prepared (conventional method) from 20 boars. Postthaw spermatozoa were used for AI, and the conception rate was assessed to categorize the animals as either good freezability (GF) or poor freezability (PF). In Experiment 2, sperm collected from three ejaculations from each boar was frozen using two separate procedures. In the first (conventional cryopreservation, control), sperm was cultured with autologous seminal plasma and held for 2 h at 15 8C. In the second (seminal plasma removed, rSP), the fresh semen was centrifuged to remove the seminal plasma, diluted in pre-treatment solution, and held for 2 h at 15 8C. The sperm in both procedures was then frozen as described above. After thawing, sperm was incubated for 1, 3 and 6 h, and then motility, capacitation and acrosomal status were determined. The oocyte fertilization rate of frozenthawed sperm was also assessed in vitro. Fresh semen or frozen-thawed spermatozoa from PF and GF boars were also used for AI. The aim of Experiment 3 was to study the low-in vivo fertility competence of frozen-thawed spermatozoa of PF boars treated with the rSP technique. The effects of the addition of seminal plasma to the thawing solution on capacitation, acrosomal status, motility and conception rate were investigated. The frozen spermatozoa of PF boars in the rSP group were thawed in Modena solution supplemented with 0, 5, 10, or 20% (v/v) seminal plasma. Forty-seven sows were inseminated using spermatozoa frozen using rSP and then thawed in the presence of 10% (v/v) seminal plasma (two-step treatment). 2.8. Statistical analysis Data were analyzed using the Statistical Analysis System Package (SAS Institute Inc., Cary, NC, USA). All percentage data of sperm assessments were subjected to arcsine transformation. The effects of boar, treatments and their interactions were analyzed by two-way ANOVA. When two-way ANOVA revealed a significant effect, means were compared using Fisher’s protected least significant difference post hoc test. The
In this study, cryopreserved spermatozoa from 20 boars were prepared by conventional methods. The motility of post-thaw spermatozoa incubated for 1 h, 3 h or 6 h differed among animals (1 h; P = 0.0001, 3 h; P = 0.003, 6 h; P = 0.0001), however there were no differences between ejaculates within each boar (1 h; P = 0.46, 3 h; P = 0.96, 6 h; P = 0.46). Based on three trials for each boar, pregnancy was not achieved with AI of frozen-thawed spermatozoa from 7 of the 20 boars. Therefore, they were defined as poor freezability boars, whereas the remaining 13 were referred to good freezability. In PF boars, the post-thawed sperm motility was lower (P < 0.05) than that of GF boars at all incubation periods (Table 1). However, there were no differences (P > 0.05) in motility among boars within same category. 3.2. Experiment 2: effects of seminal plasma during the cryopreservation process To investigate the effects of removing seminal plasma just after collection on the motility of frozenthawed spermatozoa, semen from the two different boar categories (PF and GF) was frozen by conventional cryopreservation (control) or rSP cryopreservation. No effects of rSP were observed on post-thaw motility and in vitro fertilization rate in GF boars (Table 2). However, in the PF group, there were differences (P < 0.001) between treatments, but not among the boars within same treatment group (Table 2). No interactions Table 1 Post-thaw motility rate of spermatozoa from good or poor freezability (GF and PF, respectively) boars Group
Post-thaw motility (%) Duration of incubation (h) 1
69.1 2.0 30.9 3.1
48.4 3.8 16.5 3.1
17.9 3.4 * 3.0 1.8
Values are mean S.E.M. of three ejaculates from each boar (GF; n = 13, PF; n = 7). Difference between groups (*P < 0.05, ** P < 0.01).
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Table 2 Post-thaw motility and oocyte fertilization rates of spermatozoa from good or poor freezability (GF and PF, respectively) boars frozen using control (cooling with seminal plasma and freezing without it, C), or rSP (removal of seminal plasma before cooling and freezing process) cryopreservation processes Treatment
Post-thaw motility (%)
Fertilization rate (%)
Duration of incubation (h) 1 PF C PF rSP GF C GF rSP
30.9 3.1 64.5 3.4 b 69.1 2.0 73.3 5.8
16.5 3.1 47.1 3.5 b 48.4 3.8 54.5 4.7
3.0 1.8 9.6 4.1 17.9 3.4 24.1 5.5
26.0 7.9a 74.6 6.5b 73.9 2.6 78.9 3.5
Values are mean S.E.M. of three ejaculates from each boar (GF; n = 13, PF; n = 7). (a, b) Within a column, means without a common superscript differed (P < 0.01).
between treatment and boar were detected (P > 0.05; Table 2). In the PF boars, the motility of spermatozoa in the rSP was higher (P < 0.01) than that of spermatozoa in the control at the 1- and 3-h incubation periods. Furthermore, there were differences (P < 0.01) in in vitro fertilization rates between treatment groups (26.0% vs. 74.6% for control vs. rSP). However, there was no significant effect of rSP before the freezing procedure on conception rate (PF; control 0% vs. rSP 9%) when the frozen-thawed sperm was used for AI (Table 3). To determine why the sperm lost in vivo fertilization competence in the rSP group, we analyzed the expression of a tyrosine-phosphorylated protein as a marker of capacitation, and assessed acrosomal status in frozen-thawed spermatozoa. Positive phosphotyrosine immuno-reactive bands were detected at 40 and 15 kDa. The level of expression of the 40 kDa phosphoprotein did not vary dramatically among treatments, whereas the 15 kDa phosphoprotein was highly expressed in the rSP group just after thawing (0 h) and remained high in sperm cultured for 1 h (Fig. 1A). Conversely, the 15 kDa phosphoprotein was not strongly expressed prior to culture in control sperm or in fresh sperm. In conjunction with the induction of tyrosine phosphorylation, the proportion of spermatozoa that lost their acrosome cap was increased (P < 0.01) in the rSP group compared to the control following 1-h culture (Fig. 1B). Fig. 1. Effects of the presence of seminal plasma during the freezing process on protein tyrosine phosphorylation (A) and acrosomal status (B) of frozen-thawed sperm in poor freezability (PF) boars. Sperm from PF boars was frozen in presence (control; lanes 2, 5, 8, 11) or absence of seminal plasma (rSP; lanes 3, 6, 9, 12), and these frozenthawed sperm were incubated in Modena solution without EDTA2Na. Lanes 1, 4, 7, and 10 represent fresh sperm samples (negative control). Tyrosine phosphorylation and acrosomal status were detected using an anti-phosphotyrosine antibody and FITC-PNA staining, respectively. Results in each panel were representative of three separate experiments.
Table 3 Conception rates of sows artificially inseminated with semen from poor freezability boars, fresh, or frozen-thawed, with either control (C) or removal of seminal plasma (rSP) processes Treatment
No. of sows inseminated
Conception rate (%)
Fresh AI PF C PF rSP
15 21 23
80 (12/15) 0 (0/21) 9 (2/23)
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frozen sperm from the rSP group was thawed in a solution containing 0, 5, 10 or 20% (v/v) of seminal plasma, and then cultured for up to 3 h. The addition of seminal plasma suppressed the expression of the 15 kDa phosphoprotein in a dose-dependent manner (Fig. 2A and B), with maximum effects at 10% (v/v). The presence of 10% seminal plasma in the thawing solution also decreased (P < 0.05) the rate of loss of acrosome cap (Fig. 2B). The motility of sperm was not significantly affected by the addition of seminal plasma to the thawing solution (Fig. 2C). The addition of 10% (v/v) seminal plasma during the thawing process of sperm frozen by rSP (2-step treatment group) increased (P < 0.05) the conception rate as compared to that of frozen-thawed sperm without the addition of seminal plasma to the thawing solution (2-step treatment 70% vs. rSP 9%; Table 4). The increased rate was comparable to that of fresh semen. 4. Discussion
Fig. 2. Effects of the presence of seminal plasma during the thawing process on protein tyrosine phosphorylation (A), acrosomal status (B) and motility (C) of frozen-thawed sperm from the rSP group. Frozen sperm from the rSP group was thawed in Modena solution containing varying concentrations of seminal plasma; 0 (Lanes 1, 5, 9), 5 (Lanes 2, 6, 10), 10 (Lanes 3, 7, 11), and 20% (Lanes 4, 8, 12). These spermatozoa were incubated for up to 3 h.
3.3. Experiment 3: effects of the addition of seminal plasma to the thawing solution on sperm functions In order to determine the dose effects of seminal plasma on sperm functions during the thawing process,
The presence of seminal plasma imparted resistance to cold shock in boar spermatozoa [18,19]. Indeed, the conventional method for semen cryopreservation in boars involves incubation with seminal plasma at room temperature for several hours, followed by cooling to 5 8C [20,21]. Recently, the addition of seminal plasma from good freezability boars to semen from poor freezability boars was found to improve post-thaw motility . However, Eriksson et al.  reported that extended incubation (20 h) of fresh sperm with seminal plasma decreased post-thaw sperm motility, suggesting that seminal plasma also has negative effects on sperm cryopreservation. In the present study, removal of seminal plasma before the cooling procedure did not significantly affect the motility of post-thaw sperm in good freezability boars, whereas the treatment significantly increased the motility in poor freezability animals. Although the removal of seminal plasma conflicts with the conventional freezing method described above, we propose that in PF boars, removal
Table 4 Conception rates and numbers of implantation sites in sows artificially inseminated with fresh poor freezability (PF) boar semen, or frozen-thawed spermatozoa from PF boars cryopreserved using the rSP (seminal plasma removed) or two-step treatment processes Treatment
No. of sows inseminated
Conception rate (%)
No. of implantation sites/animala
Fresh AI PF rSP PF two-step treatment*
15 23 47
80 (12/15) 9 (2/23) 70 (33/47)
12.3 0.6 9.5 1.8 12.3 0.6
Conception rate in the PF two-step treatment group was higher than that in PF rSP group (P < 0.05). a No. of implantation sites at Day 30 (both live and dead fetuses).
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of seminal plasma represents a beneficial modification of the standard boar sperm cryopreservation method. Unexpectedly, removing the seminal plasma did not significantly improve the conception rate (0 vs. 9% in control vs. rSP) in AI. The decrease of the fertilization rate in vivo was probably caused by capacitation during the thawing process, termed cryocapacitation [24,25]. Several reports have suggested that seminal plasma prevented human sperm from being capacitated, as measured by IVF  or by the fertilization of zona pellucida-free hamster oocytes . Cross  also demonstrated that the acrosomal response was greatly reduced in human ejaculated spermatozoa exposed to seminal plasma. Thus, we examined whether capacitation and acrosomal integrity in frozen-thawed spermatozoa were modified by the removal of seminal plasma before the cooling and freezing process. In the present study, the expression of a tyrosine-phosphorylated 15 kDa protein (capacitation marker) was spontaneously induced and the acrosomal cap was damaged in the frozen-thawed sperm in the rSP group. However, these effects were suppressed by the addition of seminal plasma recovered from high-reproductive performance boars to the thawing solution. Additionally, post-thaw sperm motility was not significantly influenced by additional 10% (v/v) seminal plasma. Therefore, we concluded that seminal plasma protects against spontaneous induction of the fertilization process in frozenthawed boar sperm. Perhaps the addition of seminal plasma to the thawing solution not only acts on the sperm, but also directly regulated uterine functions. Larsson  reported that when frozen sperm was thawed in the presence of seminal plasma, oocyte fertilization rates were not significantly affected, whereas the conception rate was significantly increased. Injection of seminal plasma into the female genital tract suppressed polymorphonuclear neutrophilic granulocytes in the uterus (uterine clearance) and enhanced the rate of disappearance of uterine inflammation [30–32]. Moreover, the differentiation of endometrial cells was induced by seminal plasma, and these cells expressed cytokine and chemokine mRNAs important for embryo development and implantation preparation in pigs . Thus, the addition of seminal plasma in our two-step treatment may act in part by favoring a uterine environment conducive conception and pregnancy. Further study will be required to identify which factors in seminal plasma act on sperm at each step of the cryopreservation and thawing processes. In this study, exposure to seminal plasma before the cooling and freezing process seriously damaged sperm
from poor freezability boars, whereas seminal plasma maintained oocyte penetration activity in in vivo fertilization following the thawing process. We therefore prepared frozen-thawed spermatozoa from poor freezability boars using a two-step treatment, in which seminal plasma was removed immediately after semen collection, and the cryopreserved sperm was thawed in a solution containing 10% (v/v) seminal plasma. When 47 sows were used in the subsequent insemination test, the conception rate was dramatically increased when using the frozen-thawed sperm (70%) prepared using the twostep treatment, a rate that was comparable to that using fresh semen. Therefore, we concluded that the combination of removing seminal plasma just after semen collection and the addition of 10% seminal plasma to thawing solution was a useful tool to restore in vivo fertilization competence in poor freezability boars. Acknowledgements The authors are grateful to Dr. D. Boerboom, Universite´ de Montre´al and Dr. T. Fujita, Oita Prefectural Agriculture, Forestry and Fisheries Research Center for insightful comments and suggestions concerning the manuscript. We thank the staff of the Meat Inspection Office in Oita prefecture for supplying porcine ovaries and uteri. References  Okazaki T, Abe S, Shimada M. Improved conception rates in sows inseminated with cryopreserved boar spermatozoa prepared with a more optimal combination of osmolality and glycerol in the freezing extender. Anim Sci J 2008, doi:10.1111/j.1740-0929.2008.00612.x.  Roca J, Carvajal G, Lucas X, Vazquez JM, Martinez EA. Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa. Theriogenology 2003;60:77–87.  Larsson K, Einarsson S. Influence of boars on the relationship between fertility and post thawing sperm quality of deep frozen boar spermatozoa. Acta Vet Scand 1976;17:74–82.  Hernandez M, Roca J, Gil MA, Vazquez JM, Martinez EA. Adjustments on the cryopreservation conditions reduce the incidence of boar ejaculates with poor sperm freezability. Theriogenology 2007;67:1436–45.  Johnson LA, Aalbers JG, Willems CM, Sybesma W. Use of spermatozoa for artificial insemination. I. Fertilizing capacity of fresh and frozen spermatozoa in sows on 36 farms. J Anim Sci 1981;52:1130–6.  Roca J, Hernandez M, Carvajal G, Vazquez JM, Martinez EA. Factors influencing boar sperm cryosurvival. J Anim Sci 2006;84:2692–9.  Thurston LM, Watson PF, Mileham AJ, Holt WV. Morphologically distinct sperm subpopulations defined by Fourier shape
T. Okazaki et al. / Theriogenology 71 (2009) 491–498 descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation. J Androl 2001;22: 382–94. Thurston LM, Siggins K, Mileham AJ, Watson PF, Holt WV. Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation. Biol Reprod 2002;66: 545–54. Pena FJ, Saravia F, Nunez-Martinez I, Johannisson A, Wallgren M, Rodriguez Martinez H. Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation? Anim Reprod Sci 2006;93:101–13. Rath D, Niemann H. In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars. Theriogenology 1997;47: 785–93. Kawano N, Shimada M, Terada T. Motility and penetration competence of frozen-thawed miniature pig spermatozoa are substantially altered by exposure to seminal plasma before freezing. Theriogenology 2004;61:351–64. Kikuchi K, Kashiwazaki N, Nagai T, Noguchi J, Shimada A, Takahashi R, et al. Reproduction in pigs using frozen-thawed spermatozoa from epididymis stored at 4 C. J Reprod Dev 1999;45:345–50. Vadnais ML, Kirkwood RN, Specher DJ, Chou K. Effects of extender, incubation temperature, and added seminal plasma on capacitation of cryopreserved, thawed boar sperm as determined by chlortetracycline staining. Anim Reprod Sci 2005;90:347–54. Funahashi H, Sano T. Select antioxidants improve the function of extended boar semen stored at 10 degrees C. Theriogenology 2005;63:1605–16. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. Shimada M, Yanai Y, Okazaki T, Noma N, Kawashima I, Mori T, et al. Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization. Development 2008;135:2001–11. Shimada M, Nishibori M, Isobe N, Kawano N, Terada T. Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes. Biol Reprod 2003;68:1142–9. Pursel VG, Johnson LA, Schulman LL. Effect of dilution, seminal plasma and incubation period on cold shock susceptibility of boar spermatozoa. J Anim Sci 1973;37:528–31. Tamuli MK, Watson PF. Cold resistance of live boar spermatozoa during incubation after ejaculation. Vet Rec 1994;135:60–2.
 Pursel VG, Johnson LA. Frozen boar spermatozoa: methods of thawing pellets. J Anim Sci 1976;42:927–31.  Zeng WX, Shimada M, Isobe N, Terada T. Survival of boar spermatozoa frozen in diluents of vrying osmolality. Theriogenology 2000;56:447–58.  Hernandez M, Roca J, Calvete JJ, Sanz L, Muino-Blanco T, Cebrian-Perez JA, et al. Cryosurvival and in vitro fertilizing capacity postthaw is improved when boar spermatozoa are frozen in the presence of seminal plasma from good freezer boars. J Androl 2007;28:689–97.  Eriksson BM, Vazquez JM, Martinez EA, Roca J, Lucas X, Rodriguez-Martinez H. Effects of holding time during cooling and of type of package on plasma membrane integrity, motility and in vitro oocyte penetration ability of frozen-thawed boar spermatozoa. Theriogenology 2001;55:1593–605.  Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl 2000;21:1–7.  Green CE, Watson PF. Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation. Reproduction 2001;122:889–98.  Davis B, Niwa K. Inhibition of mammalian fertilization in vitro by membrane vesicles from seminal plasma. Proc Soc Exp Biol Med 1974;146:11–6.  Kanwar KC, Yanagimachi R, Lopata A. Effects of human seminal plasma on fertilizing capacity of human spermatozoa. Fertil Steril 1979;31:321–7.  Cross NL. Multiple effects of seminal plasma on the acrosome reaction of human sperm. Mol Reprod Dev 1993;35:316–23.  Larsson K. Fertility of deep frozen boar spermatozoa at various intervals between insemination and induced ovulation: influence of boars and thawing diluents. Acta Vet Scand 1976;17:63–73.  Rozeboom KJ, Troedsson MH, Moritor TW, Crabo BG. The effect of spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts. J Anim Sci 1999;77:2201–6.  Rozeboom KJ, Troedsson MH, Hodson HH, Shurson GC, Crabo BG. The importance of seminal plasma on the fertility of subsequent artificial inseminations in swine. J Anim Sci 2000; 78:4438.  Rozeboom KJ, Rocha-Chavez G, Troedsson MH. Inhibition of neutrophil chemotaxis by pig seminal plasma in vitro: a potential method for modulating post-breeding inflammation in sows. Reproduction 2001;121:567–72.  O’Leary S, Jasper MJ, Warnes GM, Armstrong DT, Robertson SA. Seminal plasma regulates endometrial cytokine expression, leukocyte recruitment and embryo development in the pig. Reproduction 2004;128:237–47.