Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires

This study determined the relationship between two measures of field fertility of 11 high-use Australian artificial insemination (AI) dairy bulls and thirty standard laboratory assessments of spermatozoal post-thaw viability.The two measures of field fertility used, conception rates (cCR) and non-return rates (cNRR), were both corrected for all major non-bull variables. Sperm viability assessments were conducted on semen collected within the same season as that used to derive the field fertility estimates. These assessments measured sperm concentration, motility, morphology and membrane integrity at thawing, after 2h incubation and after the swim-up sperm selection procedure. Derivations of these measures and in vitro embryo fertilizing and developmental capacity were also determined. The Genstat Statistical Package [Genstat 5 Release 4.2 Reference Manual, VSN International, Oxford, 2000] was used to conduct an analysis of variance on the viability parameters across semen straws and bulls, and to calculate the strength of correlation between each semen parameter, cNRR and cCR in a correlation matrix. Step forward multiple regression identified the combination of semen parameters that were most highly correlated with cCR and with cNRR.The sperm parameters identified as being most predictive of cCR were the percentage of morphologically normal sperm immediately post-thaw (zeroNorm), the number of morphologically normal sperm after the swim-up procedure (nSuNorm), and the rate of zygote cleavage in vitro (Clv); the predictive equation formed by these parameters accounted for 70% of variance. The predictive equation produced for cNRR contained the variables zeroNorm, the proportion of membrane intact sperm after 2h incubation at 37 degrees C (twoMem) and Clv and accounted for 76.5% of the variation. ZeroNorm was found to be consistent across straws and semen batches within-bull and the sperm parameter with the strongest individual predictive capacity for both cCR (P=0.1) and cNRR (P=0.001). Post-thaw sperm parameters can be used to predict field fertility of Australian dairy sires; the calculated predictive equations are particularly useful for identifying and monitoring bulls of very high and very low potential fertility within a group.     

Sperm-lectin agglutination combined with swim-up leads to an efficient selection of highly motile, viable and heterogeneous ram spermatozoa

Lectins, high molecular weight glycoproteins with different sugar-binding specificity, are able to agglutinate different cell types. The recovery of high-quality spermatozoa can be facilitated by the agglutination induced by the lectin binding. The objective of this study was to combine sperm-lectin agglutination with a dextran/swim-up procedure for developing a new selection technique for ram spermatozoa. To study sperm quality, cell viability (plasma membrane integrity), the HOS-test response and progressive individual motility were assessed. Simultaneously, centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system was carried out to analyze sperm surface heterogeneity. Semen from 3 mature Saltz rams was pooled, and 0.5-mL aliquots were incubated with 4 fluorescein-labelled lectins (ECL, JAC, PSA, RCA). Then, a dextran solution was gently added and overlaid with medium. The top layer of the medium containing the spermatozoa was collected and replaced by careful addition of fresh medium. The incubation sequence was repeated 3 times at 10-min intervals. The consecutive 4 top layers obtained were pooled to give the swim-up combined sample. The highest rate of improvement in sperm quality was obtained after incubation with RCA, with a 50% increase in progressive individual motility, 21.6% in HOS value and 39.5% in viability. Total cell recovery was 64% (1.56x10(9) cells), with a viable cell recovery rate of 86%. The obtained sample showed 82% motility, 80% HOS score and 77% viability, up from the pre-swim-up values of 51, 60 and 57 %, respectively. Comparative CCCD analysis revealed a very high heterogeneous population in the RCA/swim-up sample obtained, while a much more homogeneous population was obtained in the sample after the dextran/swim-up procedure previously developed byus With this simple method, a large proportion of highly-motile spermatozoa with preserved plasma membrane and high heterogeneity can be obtained. These results strongly suggest that this selection procedure could result in a high fertility rate.     

Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls

The performance of frozen-thawed spermatozoa from 10 Holstein bulls in a range of in vitro diagnostic tests and the relationship with adjusted in vivo fertility data was determined. The tests included an assessment of motility (subjective and computer-assisted), morphology, concentration, viability, acrosomal and chromatin integrity conducted immediately post-thaw and after swim-up, in conjunction with membrane status (CTC staining) and migration in an artificial cervical mucus. Adjusted in vivo fertility correlated with subjectively assessed post-thaw motility (r=0.672, p=0.033), post-thaw straight-line velocity (r=0.636, p=0.048), post-thaw sperm morphology (r=-0.762, p=0.010), post-thaw sperm viability (r=0.635, p=0.048), the concentration of spermatozoa after swim-up (r=0.649, p=0.042), sperm morphology after swim-up (r=-0.687, p=0.028), the number of spermatozoa migrating 10mm into artificial cervical mucus (r=0.632, p=0.050) and the distance migrated by the vanguard spermatozoon in artificial mucus (r=0.701, p=0.024). A stepwise regression analysis identified tests which, when combined, produced models with a strong correlation (R(2)>0.9) to fertility.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20°C or cooled down to 5°C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/PI. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V, and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5°C than at 20°C, which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature.     

Relationships between the results of a modified sperm penetration test and a swim-up technique and the fertility of ram semen

Two relatively simple techniques for the objective measurement of semen quality, and which are easy to apply in the field or laboratory, have been developed: a modified sperm penetration test read by the naked eye and a colorimeter-based swim-up technique. Both the modified sperm penetration and swim-up methods produced promising results in prediction of ram fertility; the modified sperm penetration test was correlated (P<0.05) with a 48-day nonreturn rate and a 60-day conception rate; the correlation between a swim-up velocity and the 60-day conception rate approached significance (r=0.483; 0.1>P>0.05).     

Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.     

Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm

Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.     

Evaluation of fertility status of Murrah buffalo bulls in vitro using zona-free hamster eggs

Cryopreserved semen samples from 10 Murrah buffalo bulls were used for sperm penetration bioassay using zona-free hamster oocytes. The samples were evaluated for sperm motility, viability and acrosome integrity. Actively motile spermatozoa recovered by the swim-up technique were capacitated using calcium ionophore A(2 3 1 8 7). Mature female golden hamsters were superovulated with 50 IU PMSG followed 56 h later by 75 IU hCG. Cumulus mass, recovered by puncture of oviducts at the infundibulum region, was treated with 0.1% hyaluronidase and 0.1% trypsin to obtain zona-free oocytes. After coincubation of zona-free oocytes with capacitated buffalo spermatozoa, scoring was done as fertilization percentage and fertilization index. The correlation coefficients with conception rate were statistically significant with fertilization percentage (r = 0.588, P < 0.05) and fertilization index (r = 0.660, P < 0.01). However, conventional parameters like viability, motility and acrosome integrity showed poor correlation with conception rate.     

Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls

We investigated fluorometry to study sperm viability and flow cytometry to study sperm chromatin structure. We also assessed sperm quality after thawing relative to field fertility after AI as shown by 56-day non-return rates (56-d NRR) Frozen-thawed semen samples were obtained from 20 Swedish Red and White bulls (1 to 3 semen batches/bull) and the fertility data were based on 6,369 AIs. Fluorometry enabled simultaneous detection of sperm viability and concentration in Hoechst 33258-stained semen samples. Sperm chromatin structure assay (SCSA) evaluated denaturability of sperm nuclear DNA in situ after acid treatment. The intensity of fluorescence in non-permeabilized samples was negatively (r = -0.60, P < 0.001) correlated with microscopically-assessed sperm viability, and the fluorescence of permeabilized semen samples significantly (r = 0.67, P < 0.001) correlated with sperm concentration as assessed by hemocytometry. From the fluorescence output, the calculated percentage of damaged cells was negatively (r = -0.71, P < 0.001) correlated with the number of live cells derived from the microscopic assessment of sperm viability and concentration. This variable was significantly correlated with fertility results both at batch (r = -0.39, P < 0.05), and bull (r = -0.57, P < 0.01) levels. The SCSA variables SDalphat and COMPalphat were significantly (r = -0.59-0.64, P < 0.001) correlated with sperm viability variables after thawing but only the COMPalphat correlated significantly (r = -0.53, P < 0.05) with fertility results and solely at the bull level. The results indicate that fluorometric assessment is in good agreement with other practiced procedures and can be performed with sufficient accuracy. The SCSA may be a valuable complement for routinely practiced microscopic evaluation of sperm morphology of AI bull semen     

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability,chromatin structure,and field fertility

This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.     

Moribund sperm in frozen-thawed semen, and sperm motion end points post-thaw and post-swim-up, are related to fertility in Holstein AI bulls

The objectives were to compare testicular physical characteristics and post-thaw sperm characteristics and their associations with fertility in Holstein bulls used for AI. Ten Holstein bulls (4-5 y old) were classified as either high-fertility (HF) or low-fertility (LF; n = 5 each), based on adjusted 56-d non-return rates [non-return rate (NRR); range (mean ± SD): 55.6 ± 4.6 to 71.8 ± 1.3%). Testicular physical characteristics were not significantly different between the two groups. Four ejaculates were collected from each bull and cryopreserved. Several indexes of sperm motion (based on computer-assisted sperm analysis) at post-thaw and post-swim-up were correlated with NRR. Sperm from HF bulls were in transition to a hyperactivated motility pattern, whereas those from LF bulls had only a forward progressive motility pattern. In HF vs LF bulls, there was a greater percentage of viable sperm after thawing (60.6 ± 9.7 vs 49.5 ± 8.0%, P < 0.05) and after swim-up (70.9 ± 11.0 vs 63.0 ± 8.8%, P < 0.01); these two end points were positively correlated with fertility (r = 0.45, P < 0.01 and r = 0.78; P < 0.01, respectively). Furthermore, in HF vs LF bulls, the ratio of sperm recovered after swim-up to viable sperm in post-thaw semen was higher (P < 0.001), and the proportion of moribund sperm expressed as a percentage of live sperm differed (12.6 ± 3.4 vs. 16.4 ± 3.1%, P < 0.001) and was negatively correlated (r = −0.33, P < 0.05) with fertility. In conclusion, fertility of Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but it was associated with differences in moribund sperm in the inseminate, as well as characteristics of sperm post-thaw and after swim-up.     

Comparison of in vitro laboratory analyses with the fertility of cryopreserved stallion spermatozoa

Assessing the fertilizing potential of a semen sample is important for effective stallion management and for rapid progress in evaluating new cryopreservation technologies. Unfortunately, sperm motility does not estimate fertility well. These experiments established assays to measure cell viability, acrosomal integrity and mitochondrial function for cryopreserved stallion spermatozoa, using flow cytometry, and determined the variability associated with these assays. Correlations between results for these laboratory assays and stallion fertility were also determined. The inter-assay variability for visual motility, computer assisted motility, and sperm velocity, sperm viability, percent viable-acrosome intact cells and mitochondrial function of cells were all similar, however, intra-assay variability was lower for flow cytometric assays than for motility assays. The reliability of all assays were >0.72, except for sperm velocity (0.32). Although visual motility and the straightness of sperm motility conducted 90 min after thawing were correlated with seasonal fertility (0.56 and 0.55, respectively), data from no single assay were correlated with first-cycle fertility rates (P > 0.05). Best models using data from multiple assays explained 66 to 73, 76 to 89 and 79 to 94% of the variability in fertilizing potential, when two, three and four variables were included, respectively. Caution is required in interpreting these data, as only a few stallions were evaluated and relatively few mares were bred to each stallion, however, they do indicate that using a few rapid and inexpensive sperm assays, we can begin to understand factors important in stallion sperm fertilizing capacity, and we can use these assays to more effectively evaluate new methods for cryopreserving stallion spermatozoa.     

Relative efficacy of conventional sperm parameters and sperm penetration bioassay to assess bull fertility in vitro

Frozen semen samples from 10 bulls were thawed and actively motile sperm recovered using a swim-up technique. Calcium ionophore A23187 at 0.5 microM concentration was used for 1 min to induce the acrosome reaction in the sperm. Mature female golden hamsters were superovulated with 50 IU of equine chorionic gonadotrophin followed 56 h later with 75 IU of human chorionic gonadotrophin. The cumulus mass was recovered 17 h after hCG treatment by puncturing the oviducts in the infundibulum region. Subsequently, cumulus cell mass and zona pellucida were digested by 0.1% hyaluronidase and 0.1% trypsin, respectively, to yield zona-free hamster eggs (ZFE). A sperm penetration bioassay was performed by coincubating capacitated sperm at 5 X 10(6) concentration and ZFE for 3 h at 38 degrees C in an air incubator. The conception rate of the bulls was based of an average of 82.6 cows per bull with pregnancy status confirmed by rectal palpation. It was found to be strongly correlated (p < 0.01, r = 0.723) with fertilization percentage, whereas percent motile sperm, percent viable sperm and percent sperm with intact acrosomes were not significantly correlated with the conception rate (r = 0.210, -0.021 and -0.468, respectively). Results of the present study suggest that the sperm penetration bioassay can be reliably used to test the fertilizing potential of bull sperm in vitro.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

Does cleansing of frozen-thawed bull semen before assessment provide samples that relate better to potential fertility?

The present study estimated, in vitro, the influence of two cleansing methods on sperm parameters post-thaw and their relation to the fertility of the frozen-thawed semen after AI. Frozen semen from six 1-year-old Swedish Red and White dairy bulls with a range in fertility (as 56d-Non-Return Rates, i.e., 56d-NRR) of 62.2-70.7% among batches was tested, using three batches of semen per bull. From each batch, individual straws were analyzed immediately after thawing (PT, control) or pooled and subjected to a swim-up procedure (SU) or washing by centrifugation/re-suspension (W) prior to in vitro assessments. Subjective and computerized measurements of sperm motility and of concentration, morphology, and membrane integrity were recorded. SU provided spermatozoa with significantly better motility, acrosome-, midpiece- and tail morphology and membrane integrity compared to either control or W treatment. Significant, albeit low, correlations among single sperm parameters and NRR were found (after PT for tail abnormalities (r = 0.49) and average path velocity, VAP (r = 0.47), after SU for total sperm motility with CASA (r = 0.50) and after W only for non-linear motility (r = -0.69)). SU of frozen-thawed bull semen is a simple preparation procedure that selects for sperm motility and membrane integrity, essential parameters for fertilization. It helps in vitro assessment of the semen and provides a significant, although low, relationship to the fertility of the assayed semen.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: II. A fertility assay for frozen-thawed semen

Frozen-thawed sperm from five bulls with fertility rates ranging from 48% to 77% were treated with seven concentrations of dilauroylphosphatidylcholine (PC12) liposomes to induce an acrosome reaction (AR) that enabled sperm to penetrate eggs. Treated sperm were incubated with liposomes for 7 min prior to insemination of zona-free hamster eggs in vitro. Sperm and eggs were incubated 3 hr at 39°C prior to fixation, staining, and examination for sperm penetration and nuclear decondensation. The percentage of motile sperm immediately after thawing as well as after treatment with liposomes had a low correlation with sire fertility (r = .39 and ?.63, respectively). The percentage of sperm exhibiting an AR was more highly correlated with fertility (r ? ?.85). Similar correlations were found between fertility and the penetration rates of zona-free hamster eggs or the total number of penetrating sperm. When data for two high and for two lower fertility buils were each grouped to increase information per data point the correlation between the PC12 concentration giving the maximum proportion of eggs penetrated and fertility was r = .92 (P ≤ .05). The correlation between the PC12 concentration producing the most total sperm penetrating the eggs and fertility r = .97 (P ≤ .05). It was concluded that PC12 liposomes induced an AR in bull sperm frozen-thawed in egg yolk extender. Frozen-thawed sperm from low fertility bulls require less PC12 to induce the AR and to penetrate zona-free hamster eggs than do sperm from higher fertility bulls. These differences in lipid requirements may help to provide a quick, direct laboratory assay method to estimate the fertility of frozen bull semen.     

Assessment of boar semen quality in relation to fertility with special reference to methanol stress

The relationship between various semen evaluation tests and fertility in fertile and subfertile artificial insemination (AI) boars was examined. In total, 36 boars, 19 Finnish Landrace and 17 Yorkshire, were included. The average value of three ejaculates extended in an X-cell extender from each boar was used in the analysis. Based on nonreturn results (NR60d, later referred to nonreturn rate, NR%), the boars were divided into two groups: those with poor fertility (NR% < 80, n = 19) and those with normal or above average nonreturn rates (NR% = 83, n = 17). Semen quality was determined after 1 and 7 days of storage at 17 degrees C. Sperm motility before and after each methanol stress was assessed both subjectively and using a computer-assisted semen analyzer (CASA). The sperm cells were stained with calcein AM and propidium iodide and evaluated for plasma membrane integrity under an epifluorescence microscope. Propidium iodide and Hoechst 33258 dyes were used in parallel to stain sperm cells for fluorometric analysis with an automatic fluorometer. Sperm morphology was evaluated in stained smears. The percentage of sows reported as not having returned to estrus within 60 days after AI (nonreturn rate, NR%) and litter size of primiparous and multiparous farrowings were used as measures of fertility. Of the parameters analyzed, only CASA-assessed total sperm motility and methanol-stressed total sperm motility correlated significantly (P < 0.05) with nonreturn rate. Those tests presenting the highest correlation with nonreturn rate were CASA-assessed total motility (r = 0.54, P < 0.01) and subjective sperm motility (r = 0.52, P < 0.01) after 7 days of storage. The highest correlation with fertility at 1 day of storage was shown by methanol-stressed total sperm motility assessed with the CASA (r = 0.46, P < 0.01). The only semen parameter that correlated significantly (r = 0.37, P < 0.05) with litter size of multiparous farrowings was viability of seven-day stored semen stained with Hoechst 33258 and analyzed with a fluorometer. The methanol stress test described here could serve as a rapid test whose results could be used to predict NR% better than motility.     

Short-term inhibition of the energy metabolism affects motility but not surface properties of sperm cells

Centrifugal countercurrent distribution (CCCD) in aqueous two-phase systems has been proven to be a useful method to study subtle surface properties of spermatozoa. The present work shows that a short-term inhibition of the energy metabolism of sperm cells effected by incubating bovine sperm cells with KCN or ouabain, did not account for changes in the cell surface properties, as assessed either by estimation of the cell viability or by CCCD analysis. However, the short-term inhibition of energy metabolism provoked a clear decrease of cell motility, suggesting that a drop of cellular ATP levels brings about a rapid decrease of motility followed by a very delayed effect on cell surface properties. The relevance of these results on the handling of sperm and on the understanding of the molecular events underlying asthenospermia is discussed.     

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n=14 gilts) and heterospermic (n=11 gilts) insemination. Single AI's were performed under suboptimal conditions using 1 x 10(9) spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P<0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P<0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.