Fertility Of Boar Spermatozoa Research Articles

Characterization of the porcine seminal plasma proteome comparing ejaculate portions

Full identification of boar seminal plasma (SP) proteins remains challenging. This study aims to provide an extensive proteomic analysis of boar SP and to generate an accessible database of boar SP-proteome. A SP-pool (33entire ejaculates/11 boars; 3ejaculates/boar) was analyzed to characterize the boar SP-proteome. Twenty ejaculates (5 boars, 4ejaculates/boar) collected in portions (P1: first 10mL of sperm rich ejaculate fraction (SRF), P2: rest of SRF and P3: post-SRF) were analyzed to evaluate differentially expressed SP-proteins among portions. SP-samples were analyzed using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS followed by functional bioinformatics. The identified proteins were quantified from normalized LFQ intensity data. A total of 536 SP-proteins were identified, 409 of them in Sus scrofa taxonomy (374 validated with ≥99% confidence). Barely 20 of the identified SP-proteins were specifically implicated in reproductive processes, albeit other SP-proteins could be indirectly involved in functionality and fertility of boar spermatozoa. Thirty-four proteins (16 identified in S. scrofa taxonomy) were differentially expressed among ejaculate portions, 16 being over-expressed and 18 under-expressed in P1–P2 regarding to P3. This major proteome mapping of the boar SP provides a complex inventory of proteins with potential roles as sperm function- and fertility- biomarkers. Biological significanceThis proteomic study provides the major characterization of the boar SP-proteome with >250 proteins first reported. The boar SP-proteome is described so that a spectral library can be built for relative ‘label free’ protein quantification with SWATH approach. This proteomic profiling allows the creation of a publicly accessible database of the boar SP-proteome, as a first step for further understanding the role of SP-proteins in reproductive outcomes as well as for the identification of biomarkers for sperm quality and fertility.

Methods for Improving In Vitro and In Vivo Boar Sperm Fertility.

Fertility of boar spermatozoa is changed after ejaculation invivo and invitro. During processing for invitro fertilization (IVF), although spermatozoa are induced capacitation, resulting in a high penetration rate, persistent obstacle of polyspermic penetration is still observed with a high incidence. For artificial insemination (AI), we still need a large number of spermatozoa and lose a majority of those in the female reproductive tract. Fertility of cryopreserved boar spermatozoa is still injured through freezing and thawing process. In the present brief review, factors affecting fertility of boar sperm during IVF, AI and cryopreservation are discussed in the context of discovering methodologies to improve it.

Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long-term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5-SM and 15-SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5-SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5-SM and 15-SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5-SM group showed similar rates of fertilization and blastocyst formation in the fresh non-stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5-SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.

18 EFFECTS OF SKIM MILK ON THE QUALITY AND FERTILITY OF BOAR SEMEN FOLLOWING LIQUID PRESERVATION AT 5°C AND 15°C

Liquid preservation of semen can be an alternative to frozen–thawed semen for artificial insemination. The success of a selection of boar semen extenders has been studied over storage periods of 5 to 7 days. The objective of this study was to evaluate the effects of skim milk on the viability and in vitro fertility of boar spermatozoa preserved in Modena-based extenders at 5°C and 15°C for 2 weeks. A total of 7 ejaculates were collected from one boar. The sperm-rich fraction of each ejaculate was centrifuged and diluted in Modena extenders supplemented with 0 (control), 7.5, and 15 mg mL–1 of dry skim milk. The final sperm concentration was adjusted to 1 × 108 cells mL–1, and then the semen was stored at 5°C and 15°C for 2 weeks. In the first experiment, the motility, viability (live/dead fluorescence viability assay), plasma membrane integrity (hypoosmotic swelling test; HOST), and acrosome integrity (FITC-labelled peanut agglutinin staining) of semen stored for 2 weeks were assessed. In the second experiment, the fertilization of stored semen after 20 h of co-incubation with in vitro matured oocytes and their development were examined. Data were analysed using ANOVA. When the semen was stored at 5°C for 2 weeks, the mean total sperm motility of semen stored with 7.5 and 15 mg mL–1 of dry skim milk was significantly higher than that of semen in the control group (41.4% and 41.5% v. 17.4%; P &lt; 0.05). However, the beneficial effects of skim milk on the sperm motility were not observed in the semen stored at 15°C. Moreover, there were no significant differences in the other parameters of semen quality among the groups in each storage temperature. Significantly higher penetration rates of semen stored with 7.5 and 15 mg mL–1 of dry skim milk were observed in the storage at 5°C (41.1% and 34.8% v. 19.8%; P &lt; 0.05) but not at 15°C (38.9% and 26.0% v. 30.0%; P &gt; 0.05) when compared with the control group. When the semen was stored at 5°C, the development rate to the blastocyst stage of oocytes fertilized with semen stored with 7.5 mg mL–1 of dry skim milk was significantly higher than that with control and 15 mg mL–1 of dry skim milk (15.4% v. 1.1% and 7.8%; P &lt; 0.01). However, there were no significant differences in the development rates of oocytes fertilized with semen stored at 15°C among the groups (9.6–11.9%). In conclusion, our results indicate that the effect of skim milk on the viability and in vitro fertility of liquid-stored boar spermatozoa is dependent on the storage temperature. The addition of 7.5 mg mL–1 of dry skim milk may be effective for the improvement of viability and fertility of semen stored at 5°C but not at 15°C.

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.

269IN VITRO FERTILITY OF BOAR SPERMATOZOA PRESERVED AT 10^°C FOR 22 DAYS

Fertility of boar spermatozoa as determined following artificial insemination seems to be maintained during liquid preservation at 10–15°C for several days, although prolonged liquid preservations reduce the pregnancy rate rapidly. However, it is not clear if spermatozoa can penetrate into oocytes in an IVF system even after a prolonged liquid preservation. Oxidative stress could also be one of the possible detrimental factors in liquid preservation of spermatozoa. In the present study, fertility of liquid-preserved spermatozoa was examined using an IVM-IVF system. Whether cysteine can improve the fertility was also determined. Spermatozoa (from four Berkshires) was resuspended at 1×108 cells mL−1 in Modena solution containing 15% (v/v) boar seminal plasma and 0 or 5mM cysteine after washing 3 times. Sperm suspensions (1mL) were then preserved at 10°C for 22 days following a program for cooling down (to 15°C for 4h, keeping at 15°C for 12h and then to 10°C for 6h). At Days 1, 8, 15 and 22 after the start of preservation, spermatozoa (5×105 cells mL−1) were co-cultured with IVM oocytes in an IVM/IVF system (Funahashi et al., 1997 Biol Reprod 57, 49–53). Viability and functional status of spermatozoa were also examined at Days 8 and 15 of preservation by using LIVE/DEAD sperm viability kit and CTC fluorescence assay. Data (mean±SEM) from 4–6 replicates were analyzed by ANOVA and Fisher’s protected LSD test. When spermatozoa that had been preserved without cysteine (Cys−) were used, penetration rates were not different (P&amp;gt;0.05) from those with cysteine (Cys+) at Day 8 of preservation (91.4±3.4% in Cys− and 99.3±0.7% in Cys+), but lower (P&amp;lt;0.02) at Days 15 and 22 (72.6±13.6% and 33.8±8.4% in Cys−; 94.8±2.1% and 71.1±10.8% in Cys+, respectively). Both viability and proportion of uncapacitated live cells were higher (P&amp;lt;0.05) in Cys+ than Cys− at Days 8 and 15. These results demonstrate that boar spermatozoa can penetrate into oocytes in vitro even after a liquid preservation at 10°C for 22 days and that cysteine can improve the viability and penetrability in vitro of spermatozoa during liquid preservation. Supported by the Ito Foundation.

Fertility of boar spermatozoa after freezing in the absence of seminal vesicular proteins

Because exposure to seminal plasma enhances the detrimental effects of cold shock on boar spermatozoa (Lasley & Bogart, 1944; Pursel, Johnson & Rampacek, 1972), many procedures for preserving semen by deep freezing employ sperm-rich ejaculate fractions (Pursel & Johnson, 1975; Larrson & Einarsson, 1975), thereby excluding much of the seminal plasma. Our previous studies showed that a basic protein fraction of seminal vesicle origin binds irreversibly to boar spermatozoa at ejaculation (Moore & Hibbitt, 1976) and during cooling promotes sperm membrane disruption (Moore, Hall & Hibbitt, 1976). We therefore tested the possibility that boar spermatozoa could be frozen more effectively in the absence of seminal vesicular fluid.