Investigation into association and expression of PLCz and COX-2 as candidate genes for boar sperm quality and fertility

Phospholipase C zeta (PLCz) and cyclooxygenase isoenzyme type 2 (COX-2) are important in spermatogenesis, but their effect has not yet confirmed in pigs. Therefore, this study was aimed to analyse their association with sperm quality and fertility and to identify the mRNA and protein expression in boars reproductive tissues. DNA samples from 231 Pietrain (PI) and 109 Pietrain × Hampshire (PIHA) pigs with records of sperm quality [sperm concentration (SCON), motility, semen volume, plasma droplet and abnormal spermatozoa rate] and fertility (non-return rate and number of piglet born alive) traits were available. A SNP in non-coding region of PLCz g.158 A > C was associated with SCON (p < 0.05) in PIHA population while the polymorphism of COX-2 g.68 G > A in 3' UTR was not associated with any traits. For mRNA and protein expression study, a total of six boars were divided into two groups with G-I and G-II, where G-I was characterized for relatively better sperm quality. Both genes expressed higher in reproductive tissues compared with non-reproductive tissues. Phospholipase C zeta mRNA expressed higher in testis (p < 0.01), all parts of epididymis and spermatozoa from G-I, while COX-2 expressed higher in testis (p < 0.05), head and body of epididymis (p < 0.01), and spermatozoa from G-II boar. Both proteins were localized in Leydig cells and spermatozoa. These results might shed light on roles of these genes in spermatogenesis as candidate for boar sperm quality and fertility, but still the lack of association across populations should be considered.     

Investigation on association and expression of ESR2 as a candidate gene for boar sperm quality and fertility

ESR2 is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was aimed to investigate the association of ESR2 polymorphism with sperm quality and boar fertility traits and to analyse the ESR2 mRNA and protein expressions in boar reproductive tissues. DNA samples from 203 Pietrain (PI) and 100 Pietrain × Hampshire (PIHA) pigs with records of sperm quality [sperm concentration (SCON), motility (MOT), semen volume (VOL), plasma droplet rate (PDR) and abnormal spermatozoa rate (ASR)] and fertility [non-return rate (NRR) and number of piglet born alive (NBA)] traits were available. A SNP in coding region of ESR2 g.35547A>G in exon 5 was associated with MOT and PDR in the PI and with SCON, VOL, MOT and PDR in PIHA population. For mRNA and protein expression study, a total of six boars were divided into two groups with group I (G-I) and group II (G-II) where G-I characterized for relatively a better sperm quality according to the mean of two groups. mRNA expression was higher in brain and testis than that in all parts of epididymis. Both qRT-PCR and western blot analysis revealed that the ESR2 gene expression and protein expression were significantly higher in testis collected from G-II compared with that of G-I boars. Moreover, ESR2 protein localization in germ cell, Leydig and Sertoli cells, epithelial cells and spermatozoa was remarkable, which indicated the important role of ESR2 in spermatogenesis process. These results might shed new light on the roles of ESR2 in spermatogenesis as candidate for boar fertility, but still the lack of association across populations should be considered.     

Haplotype analysis of β-actin gene for its association with sperm quality and boar fertility

β‐actin (ACTB) was examined as a direct functional candidate gene for the possible association with sperm concentration, motility (MOT), semen volume per ejaculate, plasma droplet rate, abnormal sperm rate (ASR) and the fertility traits, non‐return rate and number of piglets born alive (NBA). Three polymorphisms in intron 3 (T>C) and one polymorphism in exon 4 (T>C) of porcine ACTB gene were identified by comparative sequencing of animals of the breeds Pietrain and Hampshire. Association analysis revealed that haplotypes affected the variation of the traits MOT, ASR and NBA. The beneficial haplotypes may provide considerable improvement of sperm quality and fertility in the tested commercial boar population.     

A Bayesian analysis of boar spermatozoa kinematics and head morphometrics and their relationship with litter size fertility variables

The semen movement and sperm head size patterns of boar ejaculates were analysed using computer-assisted semen analysis (CASA)-Mot and -Morph systems. The aim of the present study was to compare morphometric and kinematics variables from boars and to determine the relationship with sow fertility variables related to litter size. The females were from maternal crossing schemes such as the continuous 3-generation cross between York (Y), Landrace (L), and Pietrain (P) hybrid sows and Pietrain boars. Semen samples were collected from 11 sexually mature boars from two sire lines. Samples were analysed using the ISAS^®v1 system to evaluate eight kinematic variables of sperm velocity, progressiveness and undulations. Four morphometric parameters of sperm head size (length, width, area and perimeter) were analysed. Bayesian analysis revealed relevant differences in four kinematic variables (VSL, LIN, STR and WOB) between sire lines, with a probability of relevance (P_R) of 0.79–0.91, and Pietrain boars were associated with higher progressive motility compared with Duroc x Pietrain boars. Moreover, there were relevant differences in all morphometric variables (P_R = 0.82–0.85) between sire lines. The dam line Y-L-50 (½ Y × ½ L) had higher total born per litter and piglets born alive, and YLP-75 (¹/₈ Y × ¹/₈ L × ³/₄ P) was associated with higher values of litter weight at birth (highest posterior density region at 95% = 9.92, 16.41 kg). There are relevant differences in kinematic variables between the assessed sire lines and the differences in morphometric and litter size variables were also relevant. The York-Landrace hybrid sows had higher total born per litter and piglets born alive, and there were relevant differences when compared with YLP-50 (¼ York × ¼ Landrace × ½ Pietrain). Differences in kinematic and morphometric variables between sire and dam lines related to fertility need to be further studied.     

Motility Characteristics and Fertilizing Capacity of Boar Spermatozoa Stained with Hoechst 33342

Flow cytometry sorting of spermatozoa using fluorescence dye Hoechst 33342 is the only effective sex selection methodology validated in numerous laboratories. This study was carried out to determine the effect of Hoechst 33342 on the motility and fertility of stained boar spermatozoa. Experiment 1 evaluated motility parameters (percentage of motile spermatozoa, velocity, angularity and oscillation) of boar spermatozoa stained with Hoechst 33342 by a computer‐aided sperm analysis (CASA) instrument. Spermatozoa (30 million/ml) were divided into five treatment groups and stained during 1 h at 35°C with 9, 18, 27, 60 and 90 μM of H33342. There were no differences in sperm motility patterns nor percentages of motile spermatozoa incubated in the presence of 9, 18 or 27 μM. Percentage of motile spermatozoa and motility parameters decreased significantly (p < 0.05) at 60 μM of Hoechst 33342. Spermatozoa were immotile at concentration of 90 μM. In experiment 2, pregnancy rates, farrowing rates and litter size from sows (n = 275) artificially inseminated (AI) with either Hoechst 33342 stained (27 μM) or unstained (control) spermatozoa were determined. Sows inseminated with stained spermatozoa had no significant lower pregnancy rate (88.33%) as compared with controls (90.32%). Staining neither affected farrowing rates (85.0 vs 87.7%) nor total number of piglets born (10.56 ± 0.32 vs 10.47 ± 0.24, stained and controls, respectively). No phenotypical abnormalities were registered among the newborn piglets. The data suggest that incubating spermatozoa with Hoechst 33342 at levels required for X‐ and Y‐bearing chromosome sperm sorting, does not impair sperm viability or their fertility after AI.     

Changes in Semen Quality in Estonian Holstein AI Bulls at 3, 5 and 7 years of Age

The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.     

Porcine Field Fertility with Two Different Insemination Doses and the Effect of Sperm Morphology

In swine artificial insemination, several dose regimens are applied, ranging from 1.5 x 10(9) to 6.0 x 10(9) spermatozoa per intra-cervical insemination dose. A lower sperm dose is more profitable for artificial insemination centres and offers a more effective use of superior boars. To evaluate fertility, 50 boars were used for a total of 10 773 homospermic first inseminations at a dose of 2 billion spermatozoa. In addition, 96 boars were used at a dose of 3 billion spermatozoa for 34 789 homospermic first inseminations. Fertility was determined by a 60-day non-return rate (NR%) of first inseminations. Litter size was registered by total number of piglets born separately in primiparous and multiparous farrowings. On average, a sow was inseminated 1.5 times. A significant decrease was observed in all three fertility parameters (NR%, litter size of both primiparous and multiparous farrowings) with a dose of 2 billion spermatozoa compared with a dose of 3 billion spermatozoa. The NR% was 75.8% and 84.0% (p < 0.001), the mean litter size of primiparous farrowings 10.1 and 10.7 (p < 0.001) and the mean litter size of multiparous farrowings 11.7 and 12.1 (p < 0.001) for 2 and 3 billion spermatozoa/dose, respectively. The proportion of normal spermatozoa in the sperm morphology analysis correlated significantly with NR% in both insemination regimens: p < 0.001, r = 0.604 and p < 0.05, r = 0.223 for 2 and 3 billion spermatozoa/dose, respectively. These results confirm that quantity can at least partly compensate for poor sperm quality. When the boars with <70% normal spermatozoa in the morphology evaluation were excluded from the data there were no correlation between the sperm morphology and NR%. However, the difference between the NR% and litter size remained statistically significant (p < 0.001) in favour for the bigger insemination dose. In conclusion, a decrease in sperm dose from 3 to 2 billion spermatozoa on commercial farms will severely decrease prolificacy at least under field conditions, where a sow is inseminated an average of 1.5 times/heat, and the semen is typically used within 3 days after collection. We recommend that under commercial circumstances the homospermic semen doses contain no <3 billion spermatozoa/dose.     

Functional Sperm Parameters and Fertility of Bull Semen Extended in Biociphos-Plus® and Triladyl®

Twenty ejaculates from five dairy AI‐bulls were used to compare, in a split‐sample experiment, the fertility [56 day‐non‐return‐rate (NRR) from more than 14000 AI) and sperm viability post‐thaw of semen diluted with an egg yolk‐ (Triladyl®) or soybean‐based (Biociphos‐Plus®) commercial extender. The in vitro evaluations were divided in two experiments. Experiment 1 (n = 20) included post‐thaw evaluations of motility (subjective and computerized), membrane integrity (CalceinAM/EthD‐1, SYBR‐14/PI, and osmotic resistance test; ORT), and capacitation status (CTC/EthD‐1). Experiment 2 (n = 10) included evaluations of the capacitation‐(CTC/EthD‐1) and acrosome status (FITC‐PSA/EthD‐1) during incubation with/without a challenge with solubilized zona pellucida proteins (SZP). No significant difference in the fertility (69.1 ± 0.8 versus 69.2 ± 0.8) results was found between the two extenders. In experiment 1, the computerized motility evaluations post‐thaw (CASA) showed higher values for Biociphos‐Plus® processed semen for the velocity patterns and lateral sperm head displacement. After 6 h at room temperature (20–22°C) all the CASA motility patterns were significantly higher for Biociphos‐Plus®. The proportion of spermatozoa with intact membranes assessed by CalceinAM was significantly higher in Biociphos‐Plus® (p < 0.001) compared to Triladyl®, but such difference was not seen when using SYBR‐14 or the ORT‐assay. When using the CTC/EthD‐1 assay, a lower proportion of acrosome reacted (AR) spermatozoa post‐thaw (p < 0.01) was found in Biociphos‐Plus® processed semen, as well as a tendency (p < 0.07) for a higher number of uncapacitated spermatozoa. In experiment 2, the proportion of uncapacitated spermatozoa was significantly higher for Biociphos‐Plus® when semen was incubated (38°C and 5% CO₂) without SZP at both 0 (p < 0.001) and 30 min (p < 0.05). Concomitantly, Triladyl® showed a higher percentage of capacitated spermatozoa at 0 (p < 0.01), 30 (p < 0.05) and 120 min (p < 0.05). A higher (p < 0.05) incidence of AR‐spermatozoa was seen in Triladyl® at the beginning of the incubation with SZP. No significant difference between extenders was detected for the acrosome status by the FITC‐PSA‐assay. Incubation with SZP induced acrosome reaction of capacitated spermatozoa in both extenders, which was detected by CTC and FITC‐PSA assays. In conclusion, fertility was not affected by Biociphos‐Plus® when 15 × 10⁶ of spermatozoa per AI dose were inseminated. The finding that higher frequencies of spermatozoa seemed more membrane stable post‐thaw, when frozen in Biociphos‐Plus®, might indicate that this extender better protects the sperm viability compared with Triladyl®.     

Hyperactivation and acrosome reaction in vitro in spermatozoa ejaculated by cryptorchid dogs after orchiopexy.

Orchiopexy of the cryptorchid (CR) testis and castration of the scrotal testis were performed in three unilaterally CR beagles at six months of age. Induction rates for ejaculated sperm hyperactivation (HA) and the acrosome reaction (AR) in vitro in these orchiopexied dogs were compared with five those in normal beagles one year later. Canine spermatozoa were incubated for 9 hr at 38 degrees C under 5% CO2 in air in canine capacitation medium at a concentration of 30 x 10(6) sperm/ml. HA was observed using high-speed videomicrography. The AR spermatozoa were evaluated by the triple stain technique. As a result, there was no significant difference between 'the CR dogs after orchiopexy' (CDO) and the normal dogs (ND) with respect to sperm motility just after ejaculation. However, sperm motility of CDO decreased markedly during incubation. There was a significant difference in sperm motility between CDO (Mean +/- SD; 47 +/- 12%) and ND (80 +/- 9%) after three hours of incubation (p less than 0.01). No significant difference was observed between CDO and ND with respect to the HA rate of motile spermatozoa throughout the incubation period. The peak of HA rate was found in both CDO (58 +/- 5%) and ND (61 +/- 16%) after seven hours of incubation. The AR rate of spermatozoa in CDO was lower than that in ND after six hours of incubation. The AR rate of CDO (26 +/- 4%) was significantly lower than ND (46 +/- 5%) after eight hours of incubation (p less than 0.01). It is assumed that there might be relation between a rapid decrease of motility and low AR rate in spermatozoa of CDO during incubation.     

Cryopreservation of pig spermatozoa using carboxylated poly-L-lysine as cryoprotectant

In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.     

Motility Assessment of Porcine Spermatozoa: a Comparison of Methods

Although widely used in practice, visual motility assessment of boar spermatozoa is a very subjective method. The aim of this study was to compare the visual motility assessment of boar spermatozoa with two objective, automated systems, namely the Sperm Quality Analyzer (SQA-IIC) and the Hamilton-Thorne computer-based semen analyzer (HTR). In addition, concentrations as determined by the Burker counting chamber and HTR were compared. Motility of 30 semen samples from 30 different boars (22 Pietrain, seven Landrace boars and one Large White) was examined during three consecutive days, subjectively by two independent persons (visual motility assessment) and objectively with both automated systems. The use of the SQA-IIC and HTR for assessing boar sperm motility was evaluated and the repeatability of the measurements was estimated. The Sperm Motility Index (SMI), determined by SQA-IIC, and the percentage motile spermatozoa determined by the HTR showed a good correlation (r=0.71; p <0.05). The visual examination performed by Person 2 showed a good correlation with the SMI (r=0.81) and with the percentage of motile spermatozoa measured by the HTR (r=0.66) (p <0.05). There was a very poor correlation and a limited agreement between the visual assessments of both persons emphasizing the subjectivity of visual motility assessment. Nevertheless, motility scores of each person during the three consecutive days were highly correlated (r=0.67 and 0.72, p <0.05). The limits of agreement plots showed poor agreement between both persons and the HTR. The repeatability of measurements for most parameters evaluated by the HTR and by the SQA-IIC was good with coefficients of variation below 10%. In addition, for fertile Pietrain boars (n=22), reference values for the different HTR-parameters are presented showing a high curvilinear velocity (157.3 +/- 19.5 microm/s) and a low straightness and linearity of the movement of the spermatozoa (62.7 +/- 8.7 and 35.5 +/- 7.6%, respectively). Concentration as determined by the Burker counting chamber (56.0 +/- 16.8 x 10(6)/ml) was significantly higher compared with HTR measurement (37.6 +/- 7.7 x 10(6)/ml). The high number of counted cells and the low variation render the HTR concentration measurement more reliable. It can be concluded that visual motility assessment is highly subjective and should therefore be replaced by automated systems that allow for a more objective and detailed motility assessment of boar spermatozoa. In addition, based on the present results, highly repeatable results were obtained by the SQA-IIC and especially by the HTR.     

Validation and Usefulness of the Sperm Quality Analyzer (SQA II-C) for Bull Semen Analysis

In this study, an upgrade version of the Sperm Quality Analyzer (SQA), the SQA-IIC was tested for the assessment of bull semen quality. In Expt 1, the device showed good repeatability of measurements within and between capillaries, as evidenced by the low coefficients of variation (CVs; < 13%) at concentrations between 35 and 705 x 10(6) spermatozoa/ml. In Expt 2, 10 semen concentrations (1-1000 x 10(6)/ml) were stored in HEPES TALP for 48 h at room temperature. A time-dependent decrease in sperm motility index (SMI) values was noticed. SMI values increased linearly with increasing sperm concentrations, but remained constant around 500, corresponding to a concentration of approximately 50 x 10(6)/ml. For sperm concentrations below 50 x 10(6)/ml, SMI values were highly correlated with concentration (p < 0.05) and with semen parameters, expressing the overall semen quality (p < 0.05; Expt 3). In Expt 4, a correlation of only 0.44 (p < 0.05) between SMI values of frozen-thawed semen samples of 35 bulls and the corrected 56-day non-return rate (56dNRRc) was found. Prediction of the 56dNRRc based on the SMI value of a semen sample was inaccurate. The present study indicates that the SQA-IIC is suitable for a rapid screening of bull semen diluted to a concentration of approximately 50 x 10(6)/ml. Furthermore, the device seems inappropriate for fertility prediction.     

Effect of relaxin on the motility, acrosome reaction and utilization of glucose of fresh and frozen-thawed bovine spermatozoa

The study was conducted to investigate the effect of relaxin on motility, acrosome reaction (AR), viability and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa. Both semen samples were washed twice through centrifugation (5 min at 600 g), and preincubated for 1 h at 39°C for swim up. The swim‐up separated spermatozoa were resuspended in a sperm Tyrode's albumin lactate pyruvate (Sp‐TALP) medium containing 0 (control) and 40 ng/mL porcine relaxin and incubated for 0–6 h. Sperm motility was determined on the basis of movement quality examined by a phase contrast microscope. Sperm viability and AR were evaluated by using the triple staining technique. The incorporation and oxidation of ¹⁴C‐glucose was assessed by a liquid scintillation counter. Motility was improved (P < 0.05) in both fresh and frozen‐thawed spermatozoa by the addition of relaxin to the Sp‐TALP medium, whereas relaxin showed no significant effect on viability in either fresh or frozen‐thawed spermatozoa. The percentage of AR increased (P < 0.05) when fresh or frozen‐thawed spermatozoa were incubated with relaxin. In contrast, the incorporation and oxidation of ¹⁴C‐glucose increased (P < 0.05) in both kinds of spermatozoa incubated with relaxin. Thus the results demonstrated that the addition of relaxin to the Sp‐TALP medium increased the motility, AR and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa.     

The Effect of Oestradiol, Progesterone and Heparin on Bovine Spermatozoa Function After Thawing

The present experiment was designed to determine the effects of various biologically active substances, such as oestradiol (OE), progesterone (P4) and heparin (Hep) alone or in combination on sperm plasma membrane scrambling, capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa. Spermatozoa were incubated for 180 min in capacitation medium supplemented with (i) 1 mug/ml OE; (ii) 1 mug/ml P4; (iii) 1 mug/ml OE and 1 mug/ml P4; (iv) 1 mug/ml OE and 5 mug/ml Hep; (v) 1 mug/ml P4 and 5 mug/ml Hep; (vi) 1 mug/ml OE, 1 mug/ml P4 and 5 mug/ml Hep. At predetermined time intervals aliquots were taken to assess sperm plasma membrane scrambling, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment was aimed to study the effects of OE, P4 and OE/P4 as potential inducers of AR in Hep-capacitated spermatozoa. Plasma membrane scrambling was assessed by a flow cytometer, using Merocyanine staining. Acrosomal status and viability of spermatozoa were evaluated under epifluorescence microscope with Ethidium homodimer-1/peanut agglutinin fluorescein isothiocyanate staining method (EthD-1/PNA-FITC). The results show that OE, P4 and a combination of OE/P4 at concentrations used did not affect sperm viability. Heparin significantly (p < 0.001) increased sperm plasma membrane scrambling of OE and P4-treated spermatozoa. P4 significantly affected the rate of sperm capacitation (p < 0.001) and AR (p < 0.05), but OE expressed membrane-stabilizing properties (p < 0.05). It can be concluded that in frozen-thawed bovine spermatozoa OE presents plasma membrane stabilizing properties that can be abolished by Hep, but not by P4. Progesterone possesses capacitating and AR-inducing properties in frozen-thawed bovine spermatozoa that can be alleviated by OE.     

Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.     

Does a Boar's Season of Birth Determine Semen Parameters and Reproductive Performance?

This article studies the effect of a boar's birth season and breed on semen parameters and its further reproductive performance. Research material consisted of 72 boars from three breeds (24 Polish Large White PLW, 24 Polish Landrace PL, 24 Duroc × Pietrain D×P). During the whole period of the study, selected semen parameters were analysed: semen volume, spermatozoa concentration, total number of spermatozoa, total number of motile spermatozoa, number of insemination doses and also reproductive indicators: farrowing rate, total born litter size, total number of piglets born live and still, and average piglet weight. Boars born in the winter and summer months demonstrated the highest spermatozoa concentrations (383.25 and 392.37 × 10⁶/ml), total number of spermatozoa (91.75 and 93.21 × 10⁹), total number of motile spermatozoa (76.10 and 77.99 × 10⁹) and number of insemination doses (24.53 and 24.89; p ≤0.01). Statistically lower values for these parameters were observed for boars born in the spring and especially in autumn (p ≤0.01). The significant impact of birth season on farrowing rate (p ≤ 0.05) and average piglet weight (p ≤ 0.05) was confirmed for PLW boars. For the PL breed, only the total number of piglets born live was proven to be significantly affected (p ≤ 0.05). No impact of birth season was shown on semen quality or reproductive performance for D×P boars. In our study, we showed that the birth season of a boar had a more impact on the level of semen parameters, and less on the reproductive performance indicators. The results indicated that both the quality of semen and reproductive performance varied in terms of the study factors, as well as between individual breeds of boars involved in the experiment.     

Comparative Effects of Autologous and Homologous Seminal Plasma on the Viability of Largely Extended Boar Spermatozoa

Sperm handling, associated to artificial reproduction technologies (ART) such as in vitro fertilization (IVF) or the use of flow cytometry for cell analysis or sorting imposes volumetric extension of the sperm suspension and decreases sperm viability, presumably because of the removal of seminal plasma (SP) components. This study evaluated whether a 10% v/v of autologous SP (retrieved from the same donor boar) or homologous SP (e.g. from any of the four fertile boars included, other than the one providing the spermatozoa) would differently affect the viability of boar spermatozoa subjected to large extension in a simple saline medium [phosphate-buffered saline and 0.1% ethylenediaminetetraacetic acid (EDTA), PBSm] to a concentration of 0.3 x 10(6) spermatozoa/ml and incubated for 2 h at 30 degrees C. Sperm viability was monitored as membrane integrity [using the fluorophore carboxyfluorescein diacetate (C-FDA) and propidium iodide (PI)], mitochondrial function (using the fluorophore R-123) and motility characteristics [using Computer Assisted Sperm Analysis (CASA)]. Substraction of the SP and extension followed by incubation in PBSm significantly (p < 0.05) decreased sperm viability, which could be restored by addition of autologous SP. Furthermore, exposure of the extended spermatozoa to homologous SP (from any other individual boar) significantly (p < 0.05) varied with the source of the sire; some boars exerting beneficial effects (even surpassing the effects of the autologous SP; p < 0.05) while at least one boar negatively (p < 0.05) influencing the viability of the incubated spermatozoa. It is concluded that SP should be present when incubating highly extended spermatozoa. As a result of the obvious differences among boars, it would be advantageous to examine the ability of SP to maintain sperm viability prior to the use of SP pools during sperm handling in vitro.     

Fluorometric assessments of viability and mitochondrial status of boar spermatozoa following liquid storage

A study was conducted to assess viability and mitochondrial status of boar spermatozoa stored at 5 degrees C and 16 degrees C. Gel-free ejaculates, collected from 3 mature boars, were extended in a standard diluent (K3) supplemented with a low-density lipoprotein fraction (LDF) isolated from egg yolk, and stored for 96 h at 5 degrees C and 16 degrees C. Motility analysis was conducted after semen dilution (D0) and on D1-D4 of storage. A double staining method, rhodamine 123 (R123) and propidium iodide (PI), was used to assess sperm viability and mitochondrial status. Sperm viability was also assessed using Hoechst 33,258 (H33258) stain. In fresh semen samples, the percentage of motility was significantly correlated with the percentage of viable spermatozoa with functional mitochondria (R123-PI), viable spermatozoa determined by H33258 staining and ATP content (r = 0.88, p < or = 0.01; r = 0.69, p < or = 0.05; r = 0.77, p < or = 0.01, respectively). The ATP content was also positively correlated with the percentage of viable spermatozoa with functional mitochondria (r = 0.76, p < or = 0.01). Sperm cells progressively lost motility, viability and mitochondrial capacity when stored in the supportive media at 5 degrees C and 16 degrees C. Motility estimates were lower (p < or = 0.05) than the percentage of viable spermatozoa with functional mitochondria during storage in K3 and LDF-based diluents on D4 and D3-D4, respectively. Deterioration in motility and membrane integrity was less marked in spermatozoa stored in LDF-based diluents. Spermatozoa doubly-stained with R123-PI appeared to possess some functional mitochondria, particularly in LDF-based diluent semen. Estimates of sperm viability, as determined by R123-PI staining, were equivalent (p > or = 0.05) to estimates made using H33258 staining. A decrease in mitochondrial activity, as measured by R123 uptake, was accompanied by lower ATP content in spermatozoa stored in K3 and LDF-based diluents after 48 h and 72 h of storage, respectively. Fluorometric measurements of viability and mitochondrial status of boar spermatozoa during liquid storage seem to provide reliable information about the sperm functional membranes.     

Relationship between sperm quality traits and field-fertility of porcine semen

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 × 10⁸ sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 × 10⁶ sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 × 10⁹ spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2~76.5%) and SCI (0.16~4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity.     

Characteristics of Buck Semen with Regard to Ejaculate Numbers, Collection Intervals, Diluents and Preservation Periods

To determine the number of ejaculates which can be collected within a 20‐min period after the smallest number of days of sexual rest, and a good diluent to preserve semen for routine AI, five mature Black Bengal bucks were used in three experiments. In experiment 1, semen from the bucks were collected by using artificial vagina at homosexual mounts as many times as possible during 20 min. The ejaculate numbers 1, 3 and 4 (or 5 when the buck could produce it) were examined for important semen characteristics. The mean ejaculate volume, density, mass activity, sperm motility, sperm concentrations, total spermatozoa/ejaculate, proportion of spermatozoa with normal acrosome, midpiece and tail, and the proportion with normal head morphology varied between 267 and 342 µl, 4.1–4.5 (1–5 scale), 4.1–4.2 (1–5 scale), 77–79%, 4187 × 10⁶–5064 × 10⁶/ml, 1140 × 10⁶–1746 × 10⁶, 91–94% and 99%, respectively, depending on the collection number of the ejaculate. The difference between the ejaculates was significant only with respect to volume (p < 0.05). In experiment 2, semen was collected from the bucks successively during 20 min after 1, 2, 3 and 4 day intervals, and the first ejaculates were evaluated for the above‐mentioned semen characteristics. Semen collected after 2 or more day intervals had significantly higher volume, sperm concentration and total spermatozoa/ejaculate (p < 0.05). In experiment 3, pools of two to three ejaculates were diluted (1 : 5; semen : diluent) in splits with glucose‐citrate‐egg yolk (GCEY), Tris‐fructose‐egg yolk (TFEY) or skim milk (SM) and preserved at +4 to +7°C. Before chilling or after 0 (15 min chilling), 1, 2, 3 and 4 days of preservation, semen was evaluated for motility and proportion of normal spermatozoa with respect to acrosome, midpiece and tail. In data pooled across the bucks, the sperm motility was better in GCEY and TFEY than in SM, and the proportion of normal spermatozoa was higher in SM than in the others (p < 0.05). However, the differences in proportion of normal spermatozoa between diluents were not significant when the data were analysed separately within preservation periods. The sperm motility consistently dropped after 1 day of preservation (p < 0.01); the motility remained 50% or more up to 4 days in TFEY, 3 days in GCEY and only 2 days in SM. The proportion of spermatozoa with normal acrosome, midpiece and tail, which was generally quite high ( 90%), decreased after 3 days of preservation (p < 0.01). We conclude that Black Bengal bucks can be collected three times during 20 min, every 3 days, and that buck semen holds good motility and proportion of normal spermatozoa up to 3 days in GCEY or TFEY at 4 to 7°C.