Purification, Characterization, and Structural Studies of Proteinase Inhibitors from Boar Seminal Plasma and Boar Spermatozoa

Hyperactivity, a form of sperm motility characterized by vigorous flagellar movements, has been proposed as essential for fertilization in mammals. The objective of the present study was to establish a method for inducing hyperactivityin vitroin boar spermatozoa and to define threshold values to differentiate between hyperactive and non-hyperactive spermatozoa by computer-assisted sperm analysis (CASA) as a prerequisite for analyzing the energy metabolism during hyperactivity. In TALP-HEPES medium, non-frozen boar spermatozoa were stimulated to hyperactivity by 50 μmol l−1Ca2+within 15 min at 37 °C if 5 μmol l−1of the Ca2+ionophore A23187 was present. If 25% seminal plasma was present, boar spermatozoa required higher Ca2+concentrations (about 700 μmol l−1) for hyperactivity. Under both conditions, immobilization and head-to-head agglutination were low so that hyperactive spermatozoa could be analyzed for at least 40 min. The transition from normal to hyperactive movement was characterized by an increase in flagellar beat angle from 49° ± 12° to 200° ± 36° (n= 32) and a decrease in flagellar curvature ratio from 0.89 ± 0.04 to 0.47 ± 0.11 (n= 32). For quantification of hyperactive boar sperm, kinematic parameters of hyperactive and non-hyperactive spermatozoa were measured by CASA and statistically evaluated (receiver operating characteristic (ROC) curve analysis). The threshold values of the following four parameters were well suited for differentiating between hyperactive and non-hyperactive boar spermatozoa (ROC curve analysis: >50% specificity at 100% sensitivity). Hyperactive boar spermatozoa showed mean lateral head displacement >3.5 μm, curvilinear velocity >97 μm s−1, linearity <32% and wobble <71%. According to this multiparametric definition, induction of hyperactivity increased significantly (P< 0.0001) the fraction of hyperactive spermatozoa in semen samples from 5.1 ± 4.3% (n= 13) to 48.3 ± 6.6% (n= 7) in the absence and to 44.2 ± 7.6% (n= 10) in the presence of 25% seminal plasma, while the overall percentage of motile spermatozoa did not change significantly.

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Effect of a transcervical infusion of seminal plasma prior to insemination on the fertilising competence of low numbers of boar spermatozoa at controlled AI-ovulation intervals

Animal Reproduction Science ◽

10.1016/0378-4320(96)01553-9 ◽

1996 ◽

Vol 44 (3) ◽

pp. 165-173 ◽

Cited By ~ 6

Author(s):

D. Waberski ◽

J.A.G. Soares ◽

E. Bandeira^de Arruda ◽

K.F. Weitze

Keyword(s):

Seminal Plasma ◽

Boar Spermatozoa

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Adsorption of seminal plasma proteins by boar spermatozoa

Theriogenology ◽

10.1016/0093-691x(90)90024-n ◽

1990 ◽

Vol 34 (4) ◽

pp. 691-700 ◽

Cited By ~ 26

Author(s):

K.W. Metz ◽

Trish Berger ◽

E.D. Clegg

Keyword(s):

Seminal Plasma ◽

Plasma Proteins ◽

Seminal Plasma Proteins ◽

Boar Spermatozoa

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BIOCHEMICAL STATUS OF BOAR SPERMATOZOA and SEMINAL PLASMA BEFORE and AFTER A 10-DAY DEPLETION TEST (DT)

Reproduction in Domestic Animals ◽

10.1111/j.1439-0531.1995.tb00035.x ◽

1995 ◽

Vol 31 (1) ◽

pp. 245-246 ◽

Cited By ~ 1

Author(s):

J. Strzezek ◽

W. Demianowicz ◽

W. Kordan ◽

J. Torska ◽

P. Wysocki ◽

...

Keyword(s):

Seminal Plasma ◽

Biochemical Status ◽

Before And After ◽

Boar Spermatozoa

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

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab269 ◽

2004 ◽

Vol 16 (2) ◽

pp. 255

Author(s):

H. Funahashi

Keyword(s):

Oxidative Stress ◽

Functional Status ◽

Pregnancy Rate ◽

Seminal Plasma ◽

Artificial Insemination ◽

Fluorescence Assay ◽

Live Cells ◽

Sperm Viability ◽

Boar Spermatozoa

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>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<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<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.

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309 SEX SORTED BOAR SPERMATOZOA: TIME COURSE AND PROFILE OF THEIR IN VITRO PENETRATION ABILITY AFTER STORAGE IN THE PRESENCE OF HOMOLOGOUS SEMINAL PLASMA

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab309 ◽

2005 ◽

Vol 17 (2) ◽

pp. 305

Author(s):

I. Parrilla ◽

J.M. Vazquez ◽

M.A. Gil ◽

I. Caballero ◽

C. Almiñana ◽

...

Keyword(s):

Seminal Plasma ◽

Time Course ◽

Egg Yolk ◽

Vitro Fertilization ◽

Penetration Ability ◽

And Control ◽

Pig Oocyte ◽

Boar Spermatozoa ◽

Penetration Time

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

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

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab142 ◽

2006 ◽

Vol 18 (2) ◽

pp. 179

Author(s):

M. Sansegundo ◽

S. Ruiz ◽

A. Gonzalez ◽

N. T. Atucha ◽

C. Matás

Keyword(s):

Intracellular Calcium ◽

Seminal Plasma ◽

Gradient Method ◽

Calcium Uptake ◽

Excitation Wavelength ◽

Percoll Gradient ◽

Epididymal Spermatozoa ◽

Boar Spermatozoa ◽

Phosphate Buffered Saline

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.

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