Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen analysis

Routine semen analysis includes evaluation of concentration combined with seminal volume, morphology and motility. Subjective analysis of these parameters is known to be inaccurate, imprecise and subject to variability. Automated semen analysis could lead to an increased standardization in and between laboratories but for that to happen automated devices need to be validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended equine semen. Results were compared with computer assisted sperm analysis (CASA), which was also tested for its repeatability and agreement with LM. The SQA-Ve showed a good repeatability and fine agreement for assessing sperm concentration of raw semen based on scatter and Bland-Altman plots. This was in contrast with the motility parameters, which had a low repeatability. Morphology assessment with SQA-Ve was poorly repeatable as well as in poor agreement with LM. For extended semen, the findings were comparable. The SQA-Ve did well for concentration, whereas for the motility parameters repeatability was only just acceptable, with no agreement with LM. This sharply contrasted the CASA findings that were highly repeatable and almost in perfect agreement with LM. Based on these findings, the tested version of the SQA-Ve is insufficiently accurate to be used for analyzing raw or extended equine semen.     

Effectiveness of the sperm quality analyzer (SQA-Vp) for porcine semen analysis

The Sperm Quality Analyzer (SQA-Vp) was evaluated for assessing concentration and motility of porcine semen. Both fresh and diluted semen from 50 different boars from a commercial artificial insemination (AI) centre were investigated. For the fresh ejaculate, the concentration obtained with SQA-Vp was compared with a photometer and a haemocytometer. For the diluted samples, the concentration and motility were compared with computer assisted semen analysis (CASA) and visual sperm analysis. The agreement between methods was studied with Bland-Altman plots and the repeatability with coefficient of variation (CV) as well as Bland-Altman plots. The sperm concentration (x10⁶/ml) obtained with SQA-Vp (379.3 ± 134.9) for fresh ejaculates agreed well with concentration by the photometer (447.2 ± 154.2; difference= -67.9 x 10⁶/ml; difference + 2SD = 55.3 x 10⁶/ml; difference - 2SD = -191.1 x 10⁶/ml) and with the haemocytometer (332.8 ± 141.11; d = 92.8; d + 2SD = 448.6; d - 2SD = -263). For diluted semen, the agreement between the concentration (x10⁶/ml) assessed with SQA-Vp (20.4 ± 4.3) was good with CASA (23.2 ± 5.8; d = -2.8; d + 2SD = 6.2; d - 2 SD = -11.8) but poor with the haemocytometer (18.8 ± 5.0; d = 1.6; d+ 2SD = 12.2; d - 2SD = -9). The % motile spermatozoa assessed by SQA-Vp (65.8 ± 10.0) in diluted semen agreed well with CASA (72.2 ± 13.7; d = -6.4; d+ 2SD = 20; d - 2SD = -32.8) and with visual assessment (64.1 ± 11.6; d = 1.7; d+ 2SD = 30.9; d - 2SD = -27.5). The SQA-Vp showed a good repeatability (CV; repeatability coefficient) for measuring the concentration of both fresh (3.9%; d = 10.7; d + 2SD = 30.9; d - 2SD = -9.5) and diluted semen (2.6%; d = 1.0; d + 2SD = 2.38; d - 2SD = -0.42) and for motility (3.2%; d = 0.9; d + 2SD = 8.5; d - 2SD = -6.7). The mean SQA-Vp values fell between the other methods′ results for both fresh and diluted semen. Moreover the repeatability was acceptable. Therefore SQA-Vp can be used as a valid device for sperm quality analysis in pigs.     

Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination

In order to obtain a more standardised semen motility evaluation, Varkens KI Nederland has introduced a computer assisted semen analysis (CASA) system in all their pig AI laboratories. The repeatability of CASA was enhanced by standardising for: 1) an optimal sample temperature (39 °C); 2) an optimal dilution factor; 3) optimal mixing of semen and dilution buffer by using mechanical mixing; 4) the slide chamber depth, and together with the previous points; 5) the optimal training of technicians working with the CASA system; and 6) the use of a standard operating procedure (SOP). Once laboratory technicians were trained in using this SOP, they achieved a coefficient of variation of < 5% which was superior to the variation found when the SOP was not strictly used. Microscopic semen motility assessments by eye were subjective and not comparable to the data obtained by standardised CASA. CASA results are preferable as accurate continuous motility dates are generated rather than discrimination motility percentage increments of 10% motility as with motility estimation by laboratory technicians. The higher variability of sperm motility found with CASA and the continuous motility values allow better analysis of the relationship between semen motility characteristics and fertilising capacity. The benefits of standardised CASA for AI is discussed both with respect to estimate the correct dilution factor of the ejaculate for the production of artificial insemination (AI) doses (critical for reducing the number of sperm per AI doses) and thus to get more reliable fertility data from these AI doses in return.     

Is photometry an accurate and reliable method to assess boar semen concentration?

Sperm concentration assessment is a key point to insure appropriate sperm number per dose in species subjected to artificial insemination (AI). The aim of the present study was to evaluate the accuracy and reliability of two commercially available photometers, AccuCell™ and AccuRead™ pre-calibrated for boar semen in comparison to UltiMate™ boar version 12.3D, NucleoCounter SP100 and Thoma hemacytometer. For each type of instrument, concentration was measured on 34 boar semen samples in quadruplicate and agreement between measurements and instruments were evaluated. Accuracy for both photometers was illustrated by mean of percentage differences to the general mean. It was −0.6% and 0.5% for Accucell™ and Accuread™ respectively, no significant differences were found between instrument and mean of measurement among all equipment. Repeatability for both photometers was 1.8% and 3.2% for AccuCell™ and AccuRead™ respectively. Low differences were observed between instruments (confidence interval 3%) except when hemacytometer was used as a reference. Even though hemacytometer is considered worldwide as the gold standard, it is the more variable instrument (confidence interval 7.1%).The conclusion is that routine photometry measures of raw semen concentration are reliable, accurate and precise using AccuRead™ or AccuCell™. There are multiple steps in semen processing that can induce sperm loss and therefore increase differences between theoretical and real sperm numbers in doses. Potential biases that depend on the workflow but not on the initial photometric measure of semen concentration are discussed.     

Quality control in boar semen production by use of the FACSCount AF system

A simple and rapid flow cytometric method has recently been developed for simultaneous determination of sperm concentration and viability in semen from domestic animals. Use of SYBR-14 trade mark in combination with propidium iodide (PI) allows estimation of the proportion of live sperm (viability). An internal standard of fluorescent microspheres (beads) makes it possible to determine the sperm concentration during the same analysis. In the first experiment, the relationship between sperm viability and litter size was investigated. The second experiment explored whether a smaller variation in the number of motile sperm per insemination dose could be obtained using the FACSCount AF flow cytometer than using a spectrophotometer. Results in the first experiment show that sperm viability is closer related to litter size than is the traditionally used motility parameter. Although the flow cytometer is precise and objective, a limited effect on litter size should be anticipated if ejaculates are selected for insemination according to the percentage of viable sperm. However, the present trial used large insemination doses (2.3 x 10(9) motile sperm/dose) which partially compensate for the differences in motility and viability between boars and ejaculates. In the second experiment it was found that variation in the number of motile sperm per insemination dose could be reduced significantly if the FACSCount AF flow cytometer rather than the Corning 254 spectrophotometer was used for determination of sperm concentration in the raw semen. It is concluded that the FACSCount AF flow cytometer is a strong tool for improvement of the quality control in artificial insemination (AI) centres.     

The comparison of assessment of pigeon semen motility and sperm concentration by conventional methods and the CASA system (HTM IVOS)

The aim of these experiments was to compare conventional, microscopic methods of evaluating pigeon sperm motility and concentration to those measured by computer-assisted sperm analysis (CASA system). Semen was collected twice a week from two groups of pigeons, each of 40 males (group I: meat-type breed; group II: fancy pigeon) using the lumbo-sacral and cloacal region massage method. Ejaculates collected in each group were diluted 1:100 in BPSE solution and divided into two equal samples. One sample was examined subjectively by microscope and the second one was analysed using CASA system. The sperm concentration was measured by CASA using the anti-collision (AC) system and fluorescent staining (IDENT). There were not any significant differences between the methods of evaluation of sperm concentration. High positive correlations in both groups were observed between the sperm concentration estimated by Thom counting chamber and AC (r=0.87 and r=0.91, respectively), and between the sperm concentration evaluated by Thom counting chamber and IDENT (r=0.85 and r=0.90, respectively). The mean values for CASA measurement of proportion of motile spermatozoa (MOT) and progressive movement (PMOT) were significantly lower than the values estimated subjectively in both groups of pigeons (p< or =0.05 and p< or =0.01, respectively). Positive correlations in MOT and PMOT were noted between both methods of evaluation. The CASA system is very rapid, objective and sensitive method in detecting subtle motility characteristics as well as sperm concentration and is recommended for future research into pigeon semen.     

Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility

Two experiments were conducted to evaluate semen quality of bulls housed under controlled conditions at a large AI facility and relate results to fertility. In Experiment 1 semen was collected from six 6-yr-old bulls twice daily at 3- to 4-d intervals for 3 d. In Experiment 2 eleven 6- to 11-yr-old bulls were used. Extensive breeding information was available and semen was collected as in Experiment 1 but replicated 4 times. Standard semen analysis and computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, model 10 unit, were performed on 36 first and second ejaculates in Experiment 1 and on 44 first ejaculates in Experiment 2. Sixteen fields (2 chambers with 8 fields per chamber) were examined per sample. In Experiment 1 the correlation between estimated sperm concentration by spectrophotometry and CASA was 0.91 (P < 0.01). Among bulls the range in the percentage of motile spermatozoa was 52 to 82 for CASA versus 62 to 69 for subjective measurements made by highly experienced technicians. Thus, CASA, with high repeatability, provided a more discriminating estimate of the percentage of motile sperm cells than did the subjective procedure. Bull effect was much greater than any other variable in the experiments. Chamber differences were small and so the results for the 2 chambers with 8 fields each were combined. One to five CASA values were correlated with bull fertility, defined as 59-day nonreturn rates corrected for cow and herd effects. The percentage of motile spermatozoa accounted for a small fraction of the total variation in fertility (r2 = 0.34). However higher r2 values (0.68 to 0.98) were obtained for 2 to 5 variables used in the multiple regression equations. The results are promising, and further testing will determine more precisely which of these CASA variables are most useful in estimating bull fertility potential.     

Sperm concentration determination between hemacytometric and CASA systems: why they can be different

Determination of sperm concentration is a critical component of semen analysis. Traditionally, the hemacytometer has been the standard for calibrating other technologies used to estimate sperm concentration, including photometry, Coulter counters, flow cytometry, and computer-automated semen analysis (CASA). Disposable capillary-loaded slides are commonly used in conjunction with most CASA systems currently in use. Questions have been raised regarding differences in sperm concentration measurements between CASA systems (using 20 microm disposable slides) and hemacytometry. This review explains that these differences are largely due to the Segre-Silberberg (SS) effect, which occurs during Poiseuille flow in thin, capillary-loaded slides. The SS effect can lead to errors in estimation of particle concentration, as demonstrated with latex beads and suspensions of human or porcine spermatozoa. The SS effect does not appear to have time to develop in the hemacytometer, which at 100 microm is considerably deeper than most disposable slides. Thus, hemacytometry, when properly performed, remains the gold standard for estimation of sperm concentration. When using thin (20 microm) slides with CASA systems, recognition of the appropriate compensation factor to adjust for the SS effect is critical for accuracy.     

Computer-assisted semen analysis and its utility for profiling boar semen samples

Achieving and maintaining a successful swine AI program depends on a number of factors, including accurate semen evaluation, typically sperm motility, morphology and concentration. Computer-Assisted Semen Analysis or CASA (i.e., image analysis with a phase-contrast microscope and computer measurements of motion parameters) objectively evaluates sperm motion characteristics, morphology and concentration. A total of 3077 semen collections were evaluated with CASA (on the day of collection), and a semen dose subset was used for single-sire AI of 6266 females over 6 months. Fertility data from these inseminations were fitted with models including farm/stud, line, boar, parity, mating week, semen age at mating and boar age at mating. The residuals from these models showed no correlation for any CASA semen unique motion parameter, which could be due to the level of sperm concentration, the number of inseminations per estrus, and the low number of females mated per boar. Future studies to expand CASA/fertility analysis need to address these constraints and may include analysis of extended boar semen after storage for 1 week.     

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.     

Effects of different artificial insemination techniques and sperm doses on fertility of normal mares and mares with abnormal reproductive history

The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using fresh or frozen-thawed sperm were investigated. The material included 187 normal mares (148 foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history. Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or hysteroscopic AI onto the uterotubal junction ipsilateral to the preovulatory follicle), storage method of semen (fresh, frozen-thawed), AI volume (0.5, 2, 12 ml), and sperm dose (50 x 10(6) or 300 x 10(6) progressively motile sperm (pms) for fresh semen and 100 or 800 x 10(6) frozen-thawed sperm with >35% post-thaw motility). The mares were inseminated once per cycle, 24 h after hCG administration when fresh semen was used, or 30 h for frozen-thawed semen. Differences in pregnancy rates between treatment groups were analyzed by Chi-squared test, and for most relevant factors (insemination technique, mare, semen, and stallion) expectation values and confidence intervals were calculated using multivariate logistic models. Neither insemination technique, volume, sperm dose, nor mare or stallion had significant effects (P > 0.05) on fertility. Type of semen, breeding mares during foal heat, and an interaction between insemination technique, semen parameters, and mares did have significant effects (P < 0.05). In problem mares, frozen semen AI yielded significantly lower pregnancy rates than fresh semen AI (16/43, 37.2% versus 25/42, 59.5%), but this was not the case in normal mares. In normal mares, hysteroscopic AI with fresh semen gave significantly (P < 0.05) better pregnancy rates than uterine body AI (27/38, 71% versus 18/38, 47.3%), whereas in problem mares this resulted in significantly lower pregnancy rates than uterine body AI (5/15, 33.3% versus 16/19, 84.2%). Our results demonstrate that for problem mares, conventional insemination into the uterine body appears to be superior to hysteroscopic insemination and in normal mares, the highest pregnancy rates can be expected by hysteroscopic insemination.     

Effect of semen collection method on pre- and post-thaw Guirra ram spermatozoa

In this study, we evaluated the potential effect of the method of recovery (artificial vagina or electroejaculation) on the production and quality of Guirra ram spermatozoa cryopreserved for the possible constitution of a sperm bank. In order to address this question, we evaluated the effect of semen collection method on fresh semen quality parameters, including: volume, concentration, production, microscopic analysis (abnormal sperm and intact apical ridge) and sperm motility parameters determined by CASA system. For frozen-thawed semen, we evaluated motility parameters by CASA and intact apical ridge, acrosomal status, assessed by dual staining by IP and FITC-PNA and capacitation status, assessed by M540 and Yo-pro1, using flow cytometry. The main findings from this study were: (i) that electroejaculation resulted in a lower recovery efficiency (80% of the cases), as a consequence of contamination with urine or lack of response to the electrical stimulation; (ii) the fresh seminal quality was not significantly different between recovery methods, except for the concentration of spermatozoa, but total number of spermatozoa and the consequent number of possible seminal doses for artificial insemination were similar; and, (iii) a higher number of stable and functional spermatozoa (higher number of live non-capacitated cells, higher live acrosome intact cells and live acrosome reacted cells) were found for frozen-thawed spermatozoa collected by electro ejaculation than by artificial vagina. According to our results, we are able to develop both methodologies in the creation of the Guirra sperm bank. Assuming the advantages and limitations of both methodologies, in Guirra breed, would enable the rapid constitution of a sperm bank including samples from a large number of non-trained rams in a short period of time, which will increase the genetic variability, and so guarantee the conservation of this breed.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Validation of the sperm mobility assay in boars and stallions

The sperm mobility assay used in the present study measures the rate of sperm penetration in a biologically inert cell-separation solution (Accudenz). When a sample of sperm is overlaid in a cuvette containing Accudenz, sperm penetrate the solution and absorbance of the sample can be measured with a spectrophotometer. This assay has been successfully used to select chicken and turkey semen donors. We validated this assay for semen from boars and stallions. Absorbance was measured after overlaying fresh semen from each species in prefilled cuvettes for 1, 5, 10, 15, 20, and 40 min. There were no significant differences when sperm were incubated in prewarmed cuvettes at 37, 39, or 41 degrees C. However, a minimum concentration of 5x10(7) viable sperm/mL was needed to evaluate the rate of sperm penetration in boars. Absorbance was half-maximal at 5.4 and 14.1 min for boar and stallion sperm, respectively. Frequency analysis suggested a normal distribution of mobility values for boar sperm. There were positive correlations between mobility values and several computer-aided sperm analysis (CASA) parameters. In addition, there was medium repeatability for multiple ejaculates from single males. We concluded that the mobility assay can be used for mammalian sperm and there seemed to be phenotypic variation among boars in mobility estimates.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.     

Use of chromatin stability assay, mitochondrial stain JC-1, and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen

Cryopreservation of semen imposes deleterious effects on spermatozoa, either killing a certain proportion of cells or causing subtle damages on sperm function in the surviving population, changes not easily revealed by conventional assays. We have tested three functional assessment techniques in frozen-thawed ram semen from six adult rams, cryopreserved following eight different protocols (four extenders, and glycerol being added at two temperatures). Semen samples were thawed and the following analyses were carried out: motility (CASA), membrane integrity (Hoescht 33258 and fluorometry), chromatin status (chromatin stability test and fluorescence-assisted cell sorting, FACS) and mitochondrial activity (JC-1 and FACS). Fluorometry outcome did not correlate with the other parameters and showed large variation, albeit discriminating among cryopreservation techniques (P < 0.01). Mitochondrial activity correlated, but with low values, with total and progressive motility. However, good sperm motility and high velocity values were associated to high mitochondrial membrane potential. The chromatin stability assay was also successfully carried out, and had a good relationship with male factor (%COMP alpha(t) and SD alpha(t) parameters). In conclusion, fluorometric assessment of membrane integrity albeit rendering poor results, merits improvement, being a low-cost and handy technique, especially for work in the field. On the other hand, both assessments of chromatin stability and mitochondrial status (JC-1 staining), combined with FACS, are reliable techniques that can be used for the functional assessment of frozen-thawed ram semen.     

Influence of centrifugation or low extension rates prefreezing on the fertility of ram semen after cervical insemination

We compared the fertility of thawed ram semen, frozen according to different prefreezing semen handling protocols and previously well-defined in vitro, after cervical artificial insemination (AI) during natural estrus in Corriedale sheep. Following primary extension 1 + 1, we adjusted the final sperm concentration before packaging (200 x 10(6)/straw) either by centrifugation, in order to reconcentrate the extended semen (Protocol 1: P1), or without centrifugation, by adjusting the final sperm number by stepwise extension (Protocol 2: P2). We evaluated sperm motility (assessed both subjectively and with a computer-assisted sperm analysis instrument [CASA]), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline [CTC]) in vitro in three pooled straws of frozen-thawed semen. Three hundred Corriedale ewes, having shown spontaneous estrus during the breeding season (i.e., April, in the southern hemisphere) under extensive management conditions in Uruguay, were cervically inseminated with thawed semen from the same freezing operations as studied in vitro. The semen evaluation in vitro yielded higher percentages (P < 0.05) of damaged spermatozoa in the samples where sperm numbers were adjusted by extension before freezing (P2), compared with when adjustment was done by centrifugation (P1). However, due to the higher sperm concentration finally achieved by P2, the calculated total number of viable spermatozoa was almost equal in the two AI doses. We observed no differences in fertility between P1 and P2 for either nonreturn rates (NRRs) 21 (30.8 vs. 29.7%) and 36 (28.5 vs. 27.8%) days after AI or lambing rate (21.9 vs. 21.4%), respectively. Fertility did not differ significantly between the two different procedures of adjusting sperm numbers prior to freezing. This may indicate that the simplified protocol with adjusted extension of the semen, resulting in higher numbers of viable spermatozoa, should be the procedure of choice when freezing ram semen under field conditions. Further studies aimed at improving the modified protocol need to be performed.     

Motility characteristics of boar spermatozoa after addition of prostaglandin F2alpha

Addition of prostaglandin F2alpha (PGF2alpha) to extended boar semen has been shown to slightly increase reproductive parameters in sows such as the conception rate and the total number of piglets born alive. The mechanisms by which PGF2alpha affect these parameters have not yet been elucidated, but it is possible that the sperm transport after insemination is increased. This study investigated whether the sperm motility from 20 Piétrain boars improved when PGF2alpha (Dinolytic; 5 mg PGF2alpha/ml) was added to diluted semen. Different amounts of PGF2alpha (0, 0.5, 1 and 2 ml/100 ml) were tested and the motility was evaluated immediately after addition of PGF2alpha, after 30 min, 2 h, and 24 h. Two computer-assisted semen analysis (CASA) systems, namely the Sperm Quality Analyzer (SQA-IIC) and the Hamilton Thorne (HTR Ceros 12.1) were used to assess the motility parameters. With the SQA-IIC, sperm motility index values of the treated groups were only slightly higher (P>0.05) compared to the negative control group. The different motility parameters measured with the HTR Ceros 12.1 were similar between the treatment groups, except for beat cross frequency, which was higher in the control group (1.5-5%; P<0.001). This study documented that the addition of 2.5, 5 or 10 mg PGF2alpha to 100 ml diluted boar sperm does not increase any sperm motility parameter. Further research is necessary to elucidate mechanisms by which PGF2alpha in diluted semen may improve the reproductive performance in swine farms.     

Standardization of computer-assisted semen analysis using an e-learning application

Computer-assisted semen analysis (CASA) is primarily used to obtain accurate and objective kinetic sperm measurements. Additionally, AI centers use computer-assessed sperm concentration in the sample as a basis for calculating the number of insemination doses available from a given ejaculate. The reliability of data is often limited and results can vary even when the same CASA systems with identical settings are used. The objective of the present study was to develop a computer-based training module for standardized measurements with a CASA system and to evaluate its training effect on the quality of the assessment of sperm motility and concentration. A digital versatile disc (DVD) has been produced showing the standardization of sample preparation and analysis with the CASA system SpermVision™ version 3.0 (Minitube, Verona, WI, USA) in words, pictures, and videos, as well as the most probable sources of error. Eight test persons educated in spermatology, but with different levels of experience with the CASA system, prepared and assessed 10 aliquots from one prediluted bull ejaculate using the same CASA system and laboratory equipment before and after electronic learning (e-learning). After using the e-learning application, the coefficient of variation was reduced on average for the sperm concentration from 26.1% to 11.3% (P ≤ 0.01), and for motility from 5.8% to 3.1% (P ≤ 0.05). For five test persons, the difference in the coefficient of variation before and after use of the e-learning application was significant (P ≤ 0.05). Individual deviations of means from the group mean before e-learning were reduced compared with individual deviations from the group mean after e-learning. According to a survey, the e-learning application was highly accepted by users. In conclusion, e-learning presents an effective, efficient, and accepted tool for improvement of the precision of CASA measurements. This study provides a model for the standardization of other laboratory procedures using e-learning.