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.     

Fertility after post-cervical artificial insemination with cryopreserved sperm from boar ejaculates of good and poor freezability

This study compared the field fertility outcomes in frozen–thawed (FT) sperm from boar ejaculates with different freezability (good, GFE/poor, PFE) while testing the reliability of the post-cervical artificial insemination (post-CAI) in FT sperm. The assay was conducted over eight months with 86 weaned sows being inseminated by post-CAI. Every ejaculate in a total of 26 from 15 Piétrain boars was divided into a refrigerated semen portion (FS; control treatment) and a cryopreserved portion (FT sperm), and the ejaculates were in turn classified as GFE or PFE in function of the sperm progressive motility and viability at 240 min post-thaw. As result, one of four possible treatments was randomly given to each sow: FS-GFE, FS-PFE, FT-GFE and FT-PFE. The number of pregnant and farrowing sows in FT-GFE did not significantly differ from those of FS control treatments. Contrarily, the probabilities of pregnancy were two times lower after inseminations with FT-PFE (P < 0.05) compared to FT-GFE, which indicates that ejaculates with high post-thaw sperm progressive motility and viability are more likely to result in pregnancies than those with poor in vitro sperm function. There were no differences in litter size or the risk of backflow among treatments. Further trials are required to determine the optimal volume and concentration of FT sperm in post-CAI to obtain a more reliable method for farmers interested in cryopreserved sperm.     

The relationship between rabbit semen characteristics and reproductive performance after artificial insemination

The relationships between several rabbit buck semen traits, concerning either the ejaculate or the dose inseminated (volume, mass motility, pH, percentage of motile sperm (PMS), concentration, number of total and motile sperm per ejaculate and per insemination dose) and the reproductive performance of does was investigated in 839 inseminations involving 54 bucks and 111 does. Four genetic types were involved: INRA1601 strain (A), INRA2066 strain (B) and the two reciprocal crossbreds (AB and BA). The mating design was A x A, B x B, (AB or BA) x (AB or BA). Semen was diluted (1:9) and a constant volume of 0.5 ml was inseminated 2-4h after collection. Therefore, the total number of spermatozoa per dose was variable and proportional to the initial concentration. Mass motility significantly influenced the kindling rate. Taken separately, volume, PMS and concentration did not influence the kindling rate but their product, the number of motile sperm per ejaculate, did. Litter size (total born) was significantly influenced by concentration and all variables depending on it, particularly the number of total and motile sperm per dose. However, reproductive performances were predominantly influenced by the physiological status of the does at insemination (lactation stage and receptivity).     

Assessment of bovine sperm viability by MTT reduction assay

The MTT reduction assay depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate a simple and less costly MTT test to determine bovine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from eight bulls were included in this study. Semen sample was diluted to 30x10(6) sperms/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1h at 37 degrees C using spectrophotometer (MS2 Reader) at wave length 550nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for sperm viability, mitochondrial activity, and acrosomal integrity using SYBR-14, Rhodamine 123 and LysoTracker Green DNA-26, respectively. The correlation between the results of these tests was calculated using the Pearson correlation coefficients. The results revealed a strong correlation (P<0.001) between the results of MTT reduction rate and the results that simultaneously determined by flow cytometer, yielding correlation coefficients of r=0.950 for sperm viability, of r=0.926 for mitochondrial activity and of r=0.959 for acrosomal integrity. The same correlation coefficient was observed between the values of sperm viability calculated on the basis of MTT reduction rates and the results of flow cytometer. In conclusion, the MTT reduction test was found to be a reliable method in evaluating bovine semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.     

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Florida goats

The aims of this study were to test the presence of discrete sperm subpopulations in Florida goat ejaculates using a computer-assisted sperm analysis (CASA) system and to establish the relationship between the distribution of the subpopulations found and individual buck, total motility, and sperm concentration. Clustering methods and discriminant analysis were applied to identify motile sperm subpopulations within the semen samples. Principal component analysis revealed that three principal components represented more than the 88% of the variance. After the cluster analysis was performed four motile sperm subpopulations were identified. Subpopulation 1 consisted of rapid and linear sperm (39.84%), Subpopulation 2 consisted of slow but linear spermatozoa (33.23%), Subpopulation 3 consisted of rapid, high ALH but non-linear spermatozoa (14.63%), and Subpopulation 4 consisted of slow and non-linear spermatozoa (12.31%). There were significant differences in the distribution of the four subpopulations (P < 0.001) as well as in the percentage of total motility and the overall sperm concentration (P < 0.05) in fresh ejaculates among the four bucks tested. In conclusion, four well-defined motile sperm subpopulations were identified in Florida goat ejaculates. The relationship between the distribution of the sperm subpopulations and individual buck, total motility, and sperm concentration shows that the spermatozoa of each have different motility patterns. Therefore, the study of discrete subpopulations of motile spermatozoa could lead to a substantial increase in information acquired during caprine semen analysis.     

Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws

The fertility of frozen-thawed and fresh semen from each of three stallions was compared in an experiment with a randomized block design using 128 mares. Semen was collected every third day, extended in lactose-EDTA-egg yolk extender at a concentration of 500 × 10⁶ progressively motile sperm per 1.0 ml, and frozen in individual-dose, 1.0-ml straws (1.9 mm × 267 mm). The same stallions were collected daily for inseminations with fresh semen. For each insemination dose with fresh semen, 300 × 10⁶ progressively motile sperm were added to 10 ml of heated skim milk extender. Mares were inseminated daily from the second day of estrus through the end of estrus. Of 52 ejaculates processed and frozen, 38% were discarded because < 35% of the sperm were progressively motile after thawing. Based on rectal palpations on day 50 post-ovulation, pregnancy rates for inseminations during one estrus to semen from the three stallions were 17, 33 and 35% for frozen-thawed semen and 60, 62 and 64% for fresh semen. Pregnancy rates with frozen semen from two of the three stallions were 54% of the rates attained with fresh 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.     

Liquid storage of Asian elephant (Elephas maximus) sperm at 4 degrees C

The Asian elephant (Elephas maximus) population in the wild has been in decline for several decades and breeding in captivity has not been self-sustaining. The use of artificial insemination (AI) can help overcome many of the difficulties associated with breeding elephants in captivity; however, the ability to store semen for extended periods of time is critical to the successful application of AI to elephants. The objective of the present study was to assess the effects of four different semen extenders and the presence of egg yolk on the viability and motility of Asian elephant semen stored at 4 °C. High quality ejaculates (n=4) were collected from two Asian elephant bulls by rectal massage. Aliquots of each ejaculate were extended in four different diluents (Beltsville thawing solution (BTS); Tris–citric acid (TCA)/fructose-based; Beltsville F5 (BF5); dextrose-supplemented phosphate-buffered saline (PBS)) with or without egg yolk then cooled and stored at 4 °C. The percentages of viable (viability) and motile (motility) sperm were evaluated at 8, 24 and 48 h following collection. The addition of egg yolk significantly reduced the percentage loss in viability from initial collection to 48 h compared to extenders without egg yolk (17.0±8.2 versus 32.6±8.9 decline in percent viable sperm in the population, respectively; P<0.05). Extender and egg yolk affected (P<0.005) total motility and percent progressively motile sperm at all evaluation times during incubation. TCA + egg yolk maintained higher (P<0.05) levels of progressive motility compared to other extenders supplemented with egg yolk. These results indicate that Asian elephant semen extended in TCA diluent supplemented with egg yolk can maintain at least 50% viability and motility when stored at 4 °C for 48 h.     

Implementation of flow cytometry for quality control in four Danish bull studs

A flow cytometric method for simultaneous determination of sperm concentration and viability has recently been developed. In 2001, four Danish bull studs purchased flow cytometers and eight technicians were trained for routine analysis of raw and frozen-thawed semen. After initial training of the technicians, an experiment was carried out to document the precision of the system. The aim was also to assess if flow cytometric determination of sperm concentration could result in a more uniform production of semen doses. Results of this experiment showed high precision in the determination of sperm concentration and coefficients of variation were 3.5 and 2.4% for raw and frozen-thawed semen, respectively. Sperm viability was also assessed with high precision and coefficients of variation were 0.9% for raw semen and 1.7% for frozen-thawed semen. Furthermore, the experiment showed that package of semen doses after flow cytometric determination of sperm concentration in the raw semen results in a significantly smaller variation in the number of sperm per dose. In the second experiment, frozen semen was exchanged between the participating studs and were analysed by flow cytometry as well as by microscopic assessments by the eight technicians. Results show that the average correlation between technicians were 0.38 for motility assessments while flow cytometric agreement between technicians was significantly higher (average correlation was 0.86 for sperm viability and 0.92 for sperm concentration). The experiment also showed very high agreements between assessments within lab technician (correlations r=0.98 (sperm concentration) and r=0.99 (sperm viability)). Experiment 3 revealed that straws from the same batch varies in both concentration and viability. It is concluded that flow cytometric determination of sperm concentration and viability can be used to improve semen assessment by AI studs and result in a better quality control.     

Intra- and interboar variability in flow cytometric sperm sex sorting

To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intraboar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15 °C to 17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in three experiments. X- and Y-chromosome–bearing sperm were simultaneously separated according to the Beltsville sperm-sorting technology using a high-speed flow cytometer. In the first experiment, interboar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome–bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the receiver operating characteristics curve (0.88, P < 0.001). In the second experiment, a certain degree of intraboar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sortability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent 5 months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 hours of storage at 15 °C to 17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine artificial insemination programs.     

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.     

Centrifugation of stallion semen and its storage in large volume straws.

In a study of different methods of handling stallion semen for deep freezing, ejaculates were divided into 3 portions, the first of which was diluted 1:2 with lactose--egg yolk--glycerol diluent and frozen in pellet form. The second aliquot was centrifuged without any diluent and the third portion was initially diluted with an experimental diluent (Merck) and then centrifuged for 5 min at 1000 g. The second and third portions were frozen in large volume straws each of which contained one whole insemination dose of 1 or 2 X 10(8) progressively motile spermatozoa. The addition of a diluent to the semen before centrifugation and freezing (portion 3) resulted in an increase in sperm motility after thawing. Motility was further increased by the use of a recently developed diluent after centrifugation and before freezing. In one fertility trial, 12 of 19 mares (63%) conceived following a single insemination of frozen semen during one oestrous period.     

Fertility of fresh and frozen rabbit semen inseminated at different times is indicative of male differences in capacitation time

Some reports indicate that sperm from different males differ in capacitation time, and other reports suggest that freezing sperm may affect their capacitation time. These two variables were specifically studied in rabbits in a fertility trial with 96 does inseminated with approximately 1.6 million motile fresh or frozen sperm from three different bucks at 15, 10, 5, and 0 h before expected ovulation. Fresh semen averaged 84% live (unstained) sperm and 88% had normal acrosomes; corresponding values for frozen sperm were 44% and 54%. On the basis of does that became pregnant, average litter size with fresh semen was 5.5 and with frozen semen was 4.8 (p greater than 0.05), but overall, does bred with frozen semen produced fewer young (p less than 0.05). On the basis of total does and total semen, average litter size from insemination at 15, 10, 5, and 0 h was 2.8, 4.2, 3.8, and 1.7, and average litter size for the three bucks was 4.0, 1.8, and 3.6. There was no interaction of type of semen (fresh or frozen) with the other variables in the model (p greater than 0.05). Bucks and time of insemination affected both the proportion of does that were pregnant and litter size (p less than 0.01). A major interaction between buck and time of insemination (p less than 0.01) was due apparently to both differential sperm survival and probable capacitation time among bucks. This major interaction should be considered in designing in vitro and in vivo fertility studies, and for selecting males for use in artificial insemination.     

The relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses

The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., average: ∼50% ERR; high: ∼65% ERR).     

Increasing storage time of extended boar semen reduces sperm DNA integrity

There is an extensive use of artificial insemination (AI) in the pig industry. Extended liquid boar semen may be used for insemination for up to 5 days after collection. The objective of this study was to determine the changes in sperm quality, when boar semen was extended and stored at 18 degrees C for up to 72 h post-collection. The study included three ejaculates from five boars, for each of the four breeds: Duroc, Hampshire, Landrace and Danish Large White (n=60 ejaculates). The sperm chromatin structure assay (SCSA) showed an increase in DNA fragmentation index (DFI) after 72 h of incubation (P<0.001), with no differences between breeds (P=0.07). For two Hampshire boars, all ejaculates had a large increase in DFI after 24 h of incubation. The standard deviation of DFI (SD-DFI) differed between breeds, with the SD-DFI for Hampshire being significantly greater than for the other breeds. The SD-DFI did not change during the 72 h of storage. Sperm viability was determined using SYBR-14 and propidium iodide in combination with flow cytometry. The sperm viability did not differ between breeds (P=0.21), but a difference in viability during storage (P<0.001) was detected. In conclusion, the SCSA cytogram patterns were consistent for different ejaculates within boars and storage of extended boar semen at 18 degrees C for 72 h significantly decreased the integrity of sperm DNA.     

Effect of season on fertility of frozen buffalo semen

Twenty double ejaculates from each of ten water-buffalo bulls were collected in June (non-breeding season) and again in November (breeding season). Fresh semen was screened for sperm quantity, motility, eosin uptake, and sperm morphology and was frozen using lactose, skim-milk, and Tris extenders. Thawed semen was checked for motility and Sephadex filtration. Half of each semen batch was used for artificial insemination in the breeding season and the other half during the non-breeding season.Laboratory screening revealed that June semen had a significantly lower Sephadex filtration rate and a higher percentage of abnormal sperm cells, and three June ejaculates were excluded from further processing due to poor sperm motility. In the remaining ejaculates the motility before freezing and the sperm cell quantity were higher in June semen than in November semen. Eosin uptake, mass motility, and post-freeze-motility did not vary with season. November semen produced significantly higher pregnancy rates than June semen over a total of 3220 inseminations in both seasons. Forty percent of the observed seasonality of buffalo fertility was attributable to the male. No fertility differences appeared between extenders used. When November semen was used, the fertility in adult buffaloes in both seasons was higher than in heifers.     

Application of MTT reduction assay to evaluate equine sperm viability

The assay of MTT reduction depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate of a simple and less costly MTT test in determining equine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from 11 stallions (warm blood) were included in this study. Semen was diluted to 100 million cells/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1 and 4h at 37 degrees C using spectrophotometer (MS2 Reader) at wavelength 550 nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for mitochondrial activity, sperm viability, and acrosomal integrity using Rhodamine 123, SYBR-14 and LysoTracker Green DNA-26, respectively. The results revealed a strong correlation (P < 0.001) between the results of MTT test at 1 and 4 h of incubation time and the result of mitochondrial activity (r = 0.978, 0.977), sperm viability (r = 0.954, 0.977) and acrosomal integrity (r = 0.867, 0.886). In conclusion, the MTT test was found to be a reliable method in evaluating semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.     

Seminal traits, suitability for semen preservation and fertility in the native Portuguese horse breeds Puro Sangue Lusitano and Sorraia: Implications for stallion classification and assisted reproduction

The Puro Sangue Lusitano (PSL) is the major national breed of horse in Portugal, but no studies exist on its seminal characteristics, or on the possibility of conserving semen for future use. The aim of this study was to evaluate semen parameters, fertility and the aptness to semen preservation in Lusitano Stallions. In order to compare characteristics defined by a single or by multiple semen collections per stallion 152 ejaculates obtained from 152 Lusitano stallions presented at an annual breeding soundness examination as well as data related to 371 ejaculates obtained from 9 PSL were analyzed. These latter samples were also evaluated in terms of their possible use in assisted reproduction and were compared with 113 ejaculates obtained from 4 Sorraia horses, a rare and endangered Portuguese breed. The percentage of motile spermatozoa (PMS) was assessed after collection (AC), after semen dilution (AD) and at 24h of cool-storage. Mean values obtained for sperm motility and morphology and semen pH observed after semen collection differ significantly (P<0.05) between single collection/multiple stallions and multiple collections/limited stallions, and no age related effects were detected. Overall, Lusitano semen quality was comparable to that of related breeds, while Sorraia stallions had very poor semen quality. The response to cool-storage of diluted semen samples differed among stallions and breeds, and the best results for progressive motile sperm cells at 24h were in a range of 35-53% for PSL stallions and were lower for Sorraia stallions. Fertility rates obtained with artificial insemination (AI) averaged at 85% for PSL. With the exception of PMS AC, sperm vitality and semen pH no other seminal trait seemed to influence fertility rates in the Lusitano breed.     

Comparison of FACSCount AF system, Improved Neubauer hemocytometer, Corning 254 photometer, SpermVision, UltiMate and NucleoCounter SP-100 for determination of sperm concentration of boar semen

Current research aims at reducing the number of sperm per insemination dose thereby making measurement of sperm concentration in raw semen and the production of uniform insemination doses much more crucial. The present study evaluated the determination of sperm concentration using FACSCount AF System (FACS), Improved Neubauer hemocytometer (HEMO), Corning 254 photometer (Photo C254), SpermVision CASA System (SpermVision), UltiMate CASA System (UltiMate) and NucleoCounter SP-100 (SP-100). The instruments were evaluated with respect to repeatability and to establishing the regression curve towards both HEMO and FACS. Repeatability for the instruments was 2.7, 7.1, 10.4, 8.1, 5.4 and 3.1% for FACS, HEMO, Photo C254, SpermVision, UltiMate and SP-100, respectively. Correlation between instruments was highest between FACS and SP-100. This was made possible due to the high repeatability for both instruments. The agreement between the instruments and HEMO as the gold standard was lower than expected as the largest difference in estimation of concentration was -25 to +50%. The largest percentage difference was observed for measurements of dilute semen. It was clear that percentage difference between instruments depended on sperm concentration. In comparison to the gold standard, agreement was highest between SpermVision and HEMO for dilute semen, but for concentrated semen, agreement was highest between SP-100 and HEMO. However, the agreement between HEMO and all other instruments was not as good as expected. The reason may lie within the presence of agglutinated sperm, preventing proper HEMO counts.