Hamilton-Thorn Motility Analyzer Research Articles

Quinn’s advantage fertilization medium enhances zona pellucida-induced acrosome reaction compared with human tubal fluid medium

The aim of this study was to compare sperm velocity, hyperactivation, zona pellucida (ZP) binding and ZP-induced acrosome reaction (AR) between Quinn’s advantage fertilization (QAF), human tubal fluid (HTF) and Ham’s F10 media. Semen samples were obtained from normozoospermic men and motile spermatozoa were prepared by gradient centrifugation (PureSperm). Unfertilized oocytes from clinical IVF were used for spermatozoa–oocyte interaction tests. Sperm velocity and hyperactivation were assessed using a Hamilton-Thorn motility analyser. When media were supplemented with human albumin, sperm motility and velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF (42 ± 22 versus 21 ± 18, P < 0.001). Sperm velocity, hyperactivation and sperm binding were also significantly higher in QAF than Ham’s F10 media. Supplementation of media with either human serum or human albumin showed no difference in effect on all sperm test results. In conclusion, QAF medium significantly enhances ZP-induced AR which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF. The aim of this study was to compare sperm velocity, hyperactivated motility, sperm binding and zona pellucida (ZP)-induced acrosome reaction (AR) between Quinn’s advantage fertilization (QAF) and human tubal fluid (HTF) and Ham’s F10 media. Semen samples were obtained from men with normal semen analysis results and all test results were compared in paired control samples between different media. When media were supplemented with human albumin, sperm motility, velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF. Sperm velocity, hyperactivated motility and sperm binding were all significantly higher in QAF than Ham’s F10 media. Supplementation with either human serum or human albumin in media had no different effects on all sperm test results. In conclusion, Quinn’s advantage fertilization medium significantly enhances ZP-induced AR, which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF.

Ram Sperm Motility Parameters under The Influence of Epidermal Growth Factor

Epidermal growth factor (EGF) is one of the important cytokines that play a role in fertility. It is known that EGF affects both male and female reproduction, but its effect on sperm parameters is not fully understood. Up to the present, the effect of EGF on ram sperm motility parameters has not been published. We analyzed motility parameters of ejaculates after 24, 48, and 72 hours from the EGF addition. EGF was added to chilled ram sperm at concentrations of 0, 100, 200, and 400 ng·ml−1. Analyses were realized using computer, assisted semen analyzer (CASA)—Hamilton Thorn motility analyzer (version 7). The effect of EGF was already visible after 30 min of incubation. Significant effect on ram sperm total motility and progressive movement was observed at higher EGF concentrations after 48 h of incubation. Our results show that EGF affects sperm motility parameters depending on concentration and time of exposure.

Human sperm movement assessed with the Hamilton-Thorn motility analyzer and in vitro fertilization.

The relationship between sperm movement characteristics obtained by computerized analysis and the in vitro fertilization rates of human oocytes was studied. In 144 consecutive in vitro fertilization treatments a sample of prepared semen was analysed by a Hamilton-Thorn Motility Analyzer. In addition a visual estimation of sperm count and motility was made. Significant correlations with the fertilization rate were found for all visual parameters. Of the computerized measurements, the mean velocities of motile spermatozoa and the concentration of motile cells were significantly correlated. The average path velocity correlated best (r = 0.42, P < 0.001). There was no relationship between the percentage of motile sperm showing hyperactivated movement and the fertilization rate. A forward stepwise logistic regression analysis selected the following variables of predictive value for fertilization: average path velocity, male factor infertility as indication for in vitro fertilization, motility and concentration, as measured by the Hamilton-Thorn analyzer. A logistic regression model to predict the cases with low (< 0.2) or high fertilization rates, included the average path velocity as a significant variable and classified the samples with 90% overall accuracy. movement characteristics of spermatozoa in culture medium, especially the average path velocity are of prognostic value in prediction of human oocyte fertilization rates.

Comparitive Study of Two Computerized Semen Motility Analyzers1: Vergleichende Studie an zwei verschiedenen Computer-Samen-Analysern

Semen analysis is one of the primary tests carried out to investigate the infertile male. Subjective evaluation of semen is often prone to observer bias and error. To eliminate this, a number of computerized semen analyzers have recently been introduced into the market and we have evaluated two of the more popular models, the Cell Soft Semen Analyzer and the Hamilton Thorn Motility Analyzer (HTM 2000). The Cell Soft identifies sperm on the basis of user defined values for cell size and luminosity whereas the Hamilton Thorn identifies sperm by motility, and then applies the computer-calculated average size and luminosity of all moving objects to non moving sperm cells. Semen samples from 25 normal donors and 25 subfertile patients were analyzed using these two models of computerized semen analyzers, and also by an experienced technician using both the Makler chamber and the hemocytometer. The results obtained from the two automated analyzers were compared with those obtained by subjective evaluation. Variation in sperm count and motility were analyzed according to the sperm density. Four groups, less than 30 million/ml with debris, less than 30 million/ml, 30-50 million/ml, and greater than 50 million/ml were studied. The majority of patients fit into the first two groups. We observed that the HTM 2000 is superior to the Cell Soft in evaluating sperm count within the patient population group. For our donor population with an average sperm count of greater than 85 million/ml both systems provide extremely accurate counts.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of relaxin on motility of human sperm in vitro

Sperm of healthy men were incubated in an IVF medium with relaxin at concentrations of 3, 30, 300 and 3000 ng ml-1. Immediately after addition of relaxin and 60 and 120 min later motility, progressive motility, mean path velocity, mean progressive velocity, mean linearity and mean lateral head displacement were measured with the Hamilton-Thorn motility analyser. Neither immediately after relaxin addition, nor after 60 or 120 min, was an improvement of sperm motility observed at any concentration.

Active Immunization with Relaxin Does Not Influence Objectively Determined Sperm Motility Characteristics in Rabbits: Aktive Immunisierung mit Relaxin beeinflußt objektiv nicht die Speirnatozoen-Motilitäts-Charakteristika bei Kaninchen

Two groups of 5 rabbits were actively immunized with porcine relaxin or sham immunized. Serial analysis of sperm motion characteristics using the computer assisted Hamilton-Thorn Motility Analyzer over a period of 22 weeks did not reveal any significant antibody titers in serum and seminal plasma. In consideration of the present findings and after critical review of the literature it is concluded that an influence of relaxin on sperm motility is unlikely.

Motility and Plasma Membrane Integrity of Spermatozoa in Fractionated Stallion Ejaculates after Storage

With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with extender (Expt 1) or frozen in liquid nitrogen (Expt 2). In Expt 1, cup 1 was pre-sperm fluid, cups 2 and 3 sperm-rich fractions, and cup 4 sperm-poor fractions. In Expt 2, cups 1 and 2 were sperm-rich fractions, and cups 3 and 4 sperm-poor fractions. One sample (WE) represented the whole ejaculate in both experiments. Motility parameters were determined with a Hamilton-Thorn Motility Analyzer, and plasma membrane integrity was assessed using carboxyfluorescein diacetate and propidium iodide staining and fluorescence microscopy. The removal of seminal plasma lowered motility values, but not plasma membrane integrity, in both experiments. No significant differences between cups were observed after cooled storage. The cups differed significantly in most post-thaw motility parameters, and the sperm-rich fraction showed higher post-thaw motility than the whole ejaculate.

Hyper-osmotic condition enhances protein tyrosine phosphorylation and zona pellucida binding capacity of human sperm

The aim of this study was to determine the effect of culture medium osmolality, in the range known to occur in the male and female reproductive tracts, on human sperm tyrosine phosphorylation and sperm-zona pellucida (ZP) interaction in vitro. Motile sperm (2x10(6)), selected by swim-up from semen of normozoospermic men with normal sperm-ZP binding, were incubated with or without four oocytes in 1 ml human tubal fluid (HTF) medium with different osmolalities (150, 200, 280, 350, 400 mOsm/kg) adjusted by variation of the NaCl concentration. After 2 h incubation, the number of sperm bound to the four ZP was examined, sperm motility and velocities were assessed by Hamilton-Thorn Motility Analyzer (IVOS 10) and sperm tyrosine phosphorylation was assessed by both western immunoblotting and immunofluorescence with an anti-phosphotyrosine monoclonal antibody (PY20). The effect of hyper-osmolality (400 mOsm/kg) on the ZP-induced acrosome reaction (AR) was also determined. Incubation of human sperm in hyper-osmotic medium significantly increased tyrosine phosphorylation and the number of sperm bound to the ZP. In contrast, hypo-osmotic medium significantly decreased both tyrosine phosphorylation and sperm-ZP binding. Medium with high osmolality (400 mOsm/kg) significantly reduced the ZP-induced AR. Both hypo- and hyper-osmotic media significantly decreased average sperm percentage progressive motility and velocities. Incubation of human sperm in hyper-osmotic media was associated with significantly increased tyrosine phosphorylation and ZP-binding ability but severely reduced the ZP-induced AR.

Male Reproductive Toxicology

R.E. Chapin and J.J. Heindel, Introduction. C. Chubb, Male Mouse Sexual Behaviour Test. M.D. Culler, In Vitro Techniques for Assessing Pituitary Secretory Function. R.A. Hess and B.J Moore, Histological Methods for Evaluation of the Testis. W.F. Blazak, KA. Treinen, and P.E. Juniewicz, The Application of Testicular Spermatid Head Counts in the Assessment of Male Reproductive Toxicity. C.C. Linder and M.D. Griswold, Stage Synchronization in Rat Seminiferous Tubules Using Vitamin A Depletion and Repletion. M.L. Meistrich and Maria E.A.B. van Beek, Spermatogonial Stem Cells: Assessing Their Survival and Ability to Produce Differentiated Cells. W.M. Generoso and W. W. Piegorsch, Dominant Lethal Tests in Male and Female Mice. M. Parvinen, J. Toppari, and J. Laehdetie, Transillumination-Phase Contrast Microscopic Techniques for Evaluation of Male Germ Cell Toxicity and Matagenicity. G.R. Klinefelter, W.R. Kelce, and M.P. Hardy, The Isolation and Culture of Leydig Cells from Adult Rats. L.B. Biegel J.C. Cook, and M.E. Hurtt, Isolation and Primary Culture of Leydig Cells. A.H. Payne and L. Sha, Purification and Primary Culture of Leydig Cells. W.W. Ku and R.E. Chapin, The Preparation and Use of Sertoli Cell-Enriched Cultures from 18-Day-Old Rats. A. Steinberger and J-P. Clinton, Two-Compartment Cultures of Sertoli Cells: Applications in Testicular Toxicology. D.A. O'Brien, Isolation, Separation, and Short-Term Culture of Spermatogenic Cells. G.L. Rehnberg, The Collection of Interstitial Fluid and Seminiferous Tubule Fluid from the Rat Testis. G.R. Klinefelter, The Isolation and Culture of Epididymal Epithelial Cells from Adult Rats. B.J Danzo, Evaluation of Protein Synthesis by the Epididymis. G.P. Toth, E.J. Read, and M.K. Smith, Utilizing Cryo Resources CellSoft Computer-Assisted Sperm Analysis System for Rat Sperm Motility Studies. V.L. Slott and S. D, Perreault, Computer-Assisted Sperm Analysis of Rodent Epididymal Sperm Motility Using the Hamilton-Thorn Motility Analyzer. R. Filler, Evaluation of Rat Epididymal Sperm Morphology. J. Williams, Semen Analysis and Fertility Assessment in the Rabbit. S.M. Schrader, General Techniques for Assessing Male Reproductive Potential in Human Field Studies.

Short Communication: Lack of Breed Differences in Responses of Bovine Spermatozoa to Heat Shock

An experiment was conducted to test whether the magnitude of effects of heat shock on spermatozoal function were less for thermotolerant breeds (Brahman and other breeds with Brahman influence) than for breeds that evolved in northern Europe (Angus and Holstein). Frozen spermatozoa were thawed, purified by Percoll gradient centrifugation and incubated at 38.5, 41, or 42°C for 4h. Sperm motility was then analyzed with a Hamilton Thorn Motility Analyzer. Heat shock reduced the percentage of sperm that were motile, mean track speed, and mean path velocity. There were no significant breed × temperature interactions for these traits. The mean frequency of tail beat tended to be reduced by heat shock in bulls of Brahman-influenced breeds and, to a lesser extent, in Brahman bulls, but it was not affected by heat shock in Angus or Holstein bulls. For no traits were there significant temperature × bull within breed interactions. Overall, results indicate that 1) heat shock reduces motility of bovine spermatozoa and 2) genetic effects are unlikely to be an important determinant of the function of ejaculated sperm following heat shock.

Quality of stallion semen obtained by a new semen collection phantom (Equidame®) versus a missouri® artificial vagina

A study was performed to test a new semen collection device (Equidame® phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame® phantom with that obtained by a Missouri® artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer® transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw and chilled semen samples were cultured and the number of bacterial colonies counted after incubations of 24 and 48 h. After a 24-h incubation the number of colony-forming units (CFU) in raw semen was significantly higher (P<0.01) when collected by the Missouri® artificial vagina than by the Equidame® phantom. After cooled storage, 75% of the semen samples contained no bacteria after an incubation of 24 h, and 69% yielded no growth after 48 h. The sperm-rich fractions (Cup 2) collected by the Equidame® phantom had lower mean volumes (22.1 ± 2.3 mL [± SEM] versus 101.6 ± 9.3 mL) and significantly higher mean sperm concentrations (218.0 ± 25.8 × 10 6 vs 86.2 ± 8.1 × 10 6 cells/mL; P<0.05) than the total ejaculates collected by the Missouri® device. The total and progressive motility of chilled and frozen-thawed semen did not differ significantly between collection methods. The Equidame® phantom yielded semen that was of a lower bacteriological colony counts, but had sperm motility similar to that of semen collected with the traditional method by the Missouri® artificial vagina.

Freezing of stallion semen with addition of glycine betaine.

The effect of addition of glycine betaine to a lactose-EDTA freezing medium on the post-thaw motility of stallion semen was determined. The first three semen-rich fractions of nine stallions were collected with an open-end Krakow artificial vagina on consecutive weekdays. Semen was frozen using the Hannover method with freezing media containing glycine betaine in various concentrations from 0 to 5%. After thawing, sperm motility was analysed both by a light microscope and by a Hamilton-Thorn Motility Analyser. Total and progressive post-thaw motilities of semen containing 0.25-3% glycine betaine did not differ significantly from the total and progressive post-thaw motilities of semen frozen without glycine betaine. The total and progressive post-thaw motilities of semen containing 4 or 5% glycine betaine were significantly lower (P < 0.001) than those of semen without glycine betaine. In conclusion, glycine betaine did not show any beneficial effect on the post-thaw motility of stallion semen when semen was frozen using the Hannover method.

Aggregation of human sperm at higher temperature is due to hyperactivation.

Chemotaxis of sperm cells to chemicals and hormones, such as progesterone, helps us to understand the concept of sperm transport. Here, the hypothesis was that heat increased sperm hyperactive motility, which caused the sperm to aggregate at the higher temperature. The objectives were (1) to determine the concentration of sperm at both halves of an artificial female reproductive tract made from a hermetically sealed cryopreservation straw filled with culture medium and placed with each end at different temperatures, and (2) to analyze the motility or kinematic parameters and hyperactivation of sperm found at the different temperatures. Cryopreserved-thawed human donor sperm (N = 6) were pooled and processed through 2-layer colloid solution. Analyses of the motile sperm were carried out and the washed sperm were homogeneously mixed and pipetted into several 0.5-mL French cryopreservation straws and heat-sealed. The control substance, consisting of acid-treated sperm, was also placed in several straws. The plastic straws of sperm were placed half at 23 degrees C and half was at either 37 or 40 degrees C. After 4 h, sperm at different sections of the straws were analyzed using the Hamilton Thorn motility analyzer (HTM-C). After 4 h of incubation, the concentration of sperm was doubled at the 40 degrees C heated half of the straw when compared with the other half of the straw at 23 degrees C. There were no differences in sperm concentration in the straw kept half at 37 degrees C and half at 23 degrees C. There were significantly higher percent motility, mean average path velocity, straight line velocity, lateral head displacement, and percent hyperactivation in sperm at the 40 degrees C temperature. The aggregation of sperm at the higher temperature of 40 degrees C may be due to enhanced motility, increased sperm velocities, and a 10-fold increase in hyperactivation at that temperature. The 37 degrees C temperature was not sufficient to attract sperm. Sperm cells migrating into the higher temperature site of ovulation begin nonprogressive hyperactivation movement, which is the physiological "brake" to detain the sperm at the site of ovulation.

Effect of compatible solutes and diluent composition on the post-thaw motility of ram sperm.

The effect of the compatible solutes proline, glycine betaine and trehalose in Tris-based diluents at varying pH, concentrations of egg yolk or glycerol on the post-thaw motility characteristics and fertility of ram sperm was examined. In addition, the amino acid glycine was compared with proline, glycine betaine and a standard Tris-based diluent. Post-thaw motility was assessed using a Hamilton-Thorn motility analyser. In the presence of glycerol and egg yolk, proline and glycine betaine improved the post-thaw motility characteristics of ram sperm. Regardless of the pH of the diluent at which semen was frozen, the percentage of motile sperm was higher when frozen in the presence of proline or glycine betaine than in their absence, whereas proline and glycine betaine only improved the progressive and rapid percentages of sperm for semen frozen in diluents at pH lower than 7.0. When semen was frozen in the absence of egg yolk or glycerol all the motility characteristics were reduced. Increasing the concentration of egg yolk in the diluent from 5% to 10, 15 or 20% had no effect on the post-thaw motility of sperm. The addition of 27 mM of proline or glycine betaine to the diluent also improved post-thaw motility. However, at a concentration of 81 mM, proline and glycine betaine had a detrimental effect on the percentage of motile sperm. Trehalose had no effect on the motility of sperm frozen in glycerol-containing diluents, but motility was lower after cryopreservation in glycine than in Tris-, proline- or glycine betaine-based diluents. There were no differences in the fertility of sperm frozen in Tris-, proline or glycine betaine diluents after cervical or laparoscopic insemination of ewes.

Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa

Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75 °C and 37 °C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37 °C for 30 sec or at 75 °C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P = 0.84) between sperm motility after thawing at 37 °C and 75 °C. There was also no significant difference (P = 0.053) between the percentage of live spermatozoa using the calcein AM/ethidium homodimer stain after thawing at 37 °C and 75 °C. There was, however, a significant difference (P = 0.032) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37 °C compared with that at 75 °C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37 °C instead of 75 °C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa.

Intrauterine Fluid Accumulation in Oestrous Mares

Intrauterine fluid (IUF) was collected using a tampon from mid-oestrous mares (n = 57) with and without ultrasonically detectable accumulations of free intraluminal fluid. Bacteria were cultured and neutrophils counted from all samples (n = 57). Total protein concentration, trypsin-inhibitor capacity (TIC), and plasmin, beta-glucuronidase (B-Gase) and N-acetyl-beta-D-glucosaminidase (NAGase) activities were determined in 27 IUF samples. The motility of spermatozoa in the presence of IUF, IUF extended with Kenney's medium (1:1) and Kenney's medium alone was analysed in 9 samples using a Hamilton-Thorn motility analyser. Thirty-five mares were inseminated immediately after collection of IUF, and every second day until ovulation. Embryos were recovered nonsurgically 6 days after ovulation. After embryo transfer, fluid accumulations were recorded during oestrus and an endometrial biopsy specimen taken (n = 53). In the beginning of oestrus, fluid accumulations were detected in 39% (22/57) of mares, while on the day when IUF was collected, fluid accumulations were observed in 26% (15/57) of mares. The fluid was anechogenic, and in 80% of the mares located in the uterine body. None of the mares exhibited cytological or bacteriological evidence of acute endometritis. Total protein concentrations, TIC and B-Gase activities in IUF were statistically significantly lower in mares with fluid accumulations (n = 14) than in mares without fluid accumulations (n = 13) (p < 0.01). The addition of undiluted IUF to extended semen significantly reduced total and progressive motilities, path velocities and percentages of rapid spermatozoa (p < 0.05) in vitro. On endometrial biopsy, fibrosis was found to be more prominent (p = 0.025) in mares with fluid accumulations (n = 9) than in mares without (n = 44). It was concluded that anechogenic fluid accumulations during oestrus were associated with compositional changes in IUF. Although IUF had negative effects on spermatozoal motility in vitro, the presence of fluid accumulations at the time of insemination did not affect embryo recovery rates.

European Academy of Andrology Prize, sponsored by Hamilton Thorne Research Ltd

Summary The objective of this study was to assess the in-vitro effects of the nitric oxide substrate glyceryl trinitrate (GTN) on sperm motility and membrane lipid peroxidation. Nitric oxide (NO) can impair sperm motility, possibly by an alteration of cyclic nucleotide levels. NO may also be protective against lipid peroxidation. Semen samples from nine normospermic men were prepared by a swim-up technique. Each specimen was divided into four aliquots, one of which was the control sample. The other three had 10-6, 10-8 or 10-10 M GTN added. Sperm motility was then analysed over 180 min using a Hamilton Thorn motility analyser. Lipid peroxidation was assessed by measuring media malondialdehyde (MDA) levels at 180 min. Compared with control, the following measurements were reduced (p 0.05). The depressant effects of GTN on sperm motility appeared to be transient and reversible. The effects observed may be due to NO generated by GTN, or to GTN itself. This suggests that NO may have a role in vivo as a physiological inhibitor of sperm motility. The addition of GTN did not appear either to cause sperm membrane damage or to protect the spermatozoa from lipid peroxidation.

Effects of estrous cycle, steroids and localization of oviductal cells on in vitro secretion of sperm motility factor(s)

Experiments were performed to study the effects of day of cycle, steroids and localization of oviductal cells on the maintenance of sperm motility in vitro. Oviductal cells from cows at different stages of the estrous cycle (Days 0 to 3, 10 to 12, 18 to 20) were separated (isthmus and ampulla) and cultured for 6 d in 24 well plates with TCM-199 and 10% FCS. Medium was then replaced with TCM-199 and 6 mg/ml BSA with or without 1 μg/ml progesterone (P) or estradiol-17β (E). After 48 h, conditioned media were tested for their ability to maintain bovine sperm motility for 6 h at 37 °C using a Hamilton Thorn motility analyzer. Without steroids, sperm motility was similar with the isthmus- and ampulla-conditioned media, regardless of the stage of the cycle. With E, isthmus cell-conditioned medium from early in the cycle resulted in a significant increase in sperm motility compared with that of the controls (55% ± 10 vs 37% ± 7; P < 0.01). With P, however, the medium conditioned with ampulla cells induced a net increase in sperm motility compared to nonconditioned medium (P < 0.05). Velocity of spermatozoa was also higher in conditioned media (± steroids; 123μm/sec ± 7) than that of controls (57 μm/sec ± 0.5; P < 0.0001). In summary, oviductal cells stimulate survival and motility of bovine sperm in vitro in a hormone-dependent manner.

Evaluation of a modified sperm penetration test in ram semen

A modified sperm penetration test, based on the movement of spermatozoa into a capillary tube containing 300 mM Tris-28 mM glucose (TG) solution, was developed for evaluating ram semen. Sperm penetration distance increased from 4.8 mm at 1 × 10 9 sperm/ml to 7.4 mm at 2.5 × 10 9 sperm/ml (P < 0.01) and, with time, from 3.5 at 5 min to 8.1 at 20 min (P < 0.001). A semen concentration of 2.0 × 10 9 sperm/ml (diluted as necessary with TG) and a penetration time of 15 min (a compromise between high sperm penetration distance and speed of operation) are recommended for routine tests. Sperm penetration distance was validated by the fact that it was highly correlated to both the known proportions of motile spermatozoa in the test samples (r = 0.76 to 0.90; P < 0.001) and, in the aged, cold-shocked, diluted-stored, and frozen-thawed semen samples to most parameters from a computerized Hamilton Thorn Motility Analyzer (r = 0.51 to 0.93; P < 0 05 to < 0.001). The modified sperm penetration test described here is thus a useful addition to the means available for evaluating ram semen. The low cost of the test and the fact that it does not require electric power as well as and the availability and nature of Tris and glucose make the technique particularly useful for field work and in developing countries.

Distribution of variance associated with measurement of post-thaw function in ram sperm

Post-thaw characteristics of ram semen frozen as pellets were assessed using biochemically (amidase activity) or motility-based (Hamilton Thorn Motility Analyzer) techniques. The total variation associated with each semen characteristic measured was partitioned between rams (5), ejaculates within rams (5), pellets within ejaculates (5) and within pellets (2). A variety of variance distributions were observed for the characteristics measured. Of the 18 post-thaw characteristics examined, 10 had > 50% of variance distributed between within-ejaculate components. This has important implications for the way in which such measurements may be used in post-thaw semen analysis.