Normal and abnormal response to sperm deposition in female dogs: A review and new hypotheses for endometritis.

In mammalian species there are significant physiological responses of the female reproductive tract to the deposition of sperm. These are particularly notable in species where sperm are deposited directly into the uterus, and function both to facilitate sperm transport to the sperm reservoir, and to eliminate introduced contaminants. In the bitch, sperm are deposited into the vagina and are rapidly transported through the open cervix. Sperm are then distributed around the uterus by uterine contractions such that transportation to the tip of the uterine horns occurs within 1 min of the start of mating. The main sperm reservoir appears to be the distal part of the utero-tubal junction which forms a pre-uterine tube reservoir. Sperm remain attached here by their heads to uterine epithelium and remain viable. In non-capacitating conditions sperm slowly detach from this site and this seems important to replenish the uterine tube reservoir, where sperm may re-attach to the epithelium. Post-ovulatory signals trigger capacitation changes and subsequent hyperactivated motility that is associated with detachment of sperm from both reservoirs; thus facilitating fertilization. After mating, a physiological post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils, increased uterine contractions, an increased uterine artery blood flow and a decrease of the resistance index indicating a short-duration vasodilation. Disturbance of this tightly regulated system has the potential to impact fertility by a failure of elimination of the introduced contaminants (such that a clinically-significant post-breeding endometritis ensues) but also by impairing sperm transport.

Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm.

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2-3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.

Current knowledge on the transport and fate of spermatozoa in the reproductive tract of the bitch.

Canine sperm transport, distribution, storage and detachment is a complex, dynamic and highly regulated process. Transport of sperm within the bitch's reproductive tract is rapid and is influenced by the method of semen deposition (natural mating or artificial insemination) and by the timing of breeding in relation to the day of ovulation. The fertile lifespan of spermatozoa in the reproductive tract of the bitch is considerably longer than in most other domestic species, and the main sperm reservoirs appear to be the uterine crypts and the distal part of the uterotubal junction, where spermatozoa attach by their heads to uterine epithelium. While several in vitro studies demonstrated prolonged motility and viability of canine spermatozoa after coincubation with uterine tube explants, spermatozoal storage has not been documented in the canine uterine tube isthmus or ampulla in vivo. Several factors, including exposure to progesterone, solubilized zona pellucida proteins and post-ovulation uterine tube fluid, appear to trigger membrane events resulting in capacitation-like changes with subsequent motility pattern changes (transitional and hyperactivated) that are associated with sperm detachment. After mating or insemination, a normal low-magnitude post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils (PMNs), increased uterine contractions and an increased uterine artery blood flow. Recently, it was also shown that normal dogs with cystic endometrial hyperplasia develop a more significant endometritis, show fewer mating-induced uterine contractions, a decreased ability of spermatozoa to bind to uterine explants in vitro and a slower uterine clearance after mating.

Cats and dogs: two neglected species in this era of embryo production in vitro?

During the last decades, in vitro fertilization (IVF) has become a routine technique in most domestic animals. However, in the dog the technique has lagged behind, with to date not a single pup born after IVF. In cats, healthy kittens have been born, but in fewer numbers than in cattle and horses. In pet animals, research in reproduction has mainly been focused on contraception, although recently, the introduction of new drugs especially marketed for cats and dogs will probably expand fertility research in carnivores towards the previously neglected area of assisted reproduction. In particular, the dog remains a real challenge for the reproductive biologist, due to the low meiotic capacity of canine follicular oocytes. In cats, oocyte maturation is less of a problem and embryo production rates comparable to those of cattle can be achieved. The domestic cat is a valuable model for endangered felids and it can even be used as a recipient for wild felid embryos. In this short review, we list some of the problems associated with the implementation of IVF in dogs and cats in relation to their reproductive characteristics, and we discuss the state-of-the-art of IVF in several other domestic species such as cattle, horses and pigs.

Reproduction in the endangered African wild dog: basic physiology, reproductive suppression and possible benefits of artificial insemination.

The African wild dog (Lycaon pictus) is an endangered exotic canid with less than 5500 animals remaining in the wild. Despite numerous strategies to conserve this species, numbers of free-living animals are in decline. It is a highly social species with a complex pack structure: separate male and female dominant hierarchies with, typically, participation of subdominant adults in the rearing of the dominant breeding pairs' pups. Basic reproductive knowledge is largely missing in this species, with only limited information available on the profile of reproductive hormones, based on non-invasive endocrine monitoring. The dominant or alpha male and female are reproductively active and the subdominants are generally reproductively suppressed. However, the occasional production of litters by subdominant females and evidence of multiple paternity within litters suggests that fertility of subordinates is not completely inhibited. In this respect, there are still considerable gaps in our knowledge about the mechanisms governing reproduction and reproductive suppression in African wild dogs, particularly the influence of dominance and pack structure on both male and female fertility. Given concerns over the long-term survival of this species, further research in this area is essential to provide valuable information for their captive breeding and conservation. Reproductive information can also be applied to the development of Assisted Reproductive Techniques for this species; the utility of which in African wild dog conservation is also discussed.

Computer-assisted sperm analysis in dogs and cats: an update after 20 years.

In dogs and cats, computer-assisted sperm analysis (CASA) was originally described almost 20 years ago. Subsequently, numerous CASA systems were validated and used for various applications in dogs and to a lesser extent in cats. CASA systems offer an accurate, rapid, objective and simultaneous assessment of different semen parameters allowing the visualization of subtle changes in sperm characteristics, which cannot be identified by conventional semen analysis. The main problems of these computerized measuring devices are the relatively high investment costs and the need for standardization and validation before any practical use is possible. In comparison with automated motility and concentration assessment, automated morphometry and morphology assessment is more complex and time-consuming. Once validated, CASA systems can be routinely used in veterinary centres for assessment of fertility and for the improvement of sperm diluters, cooling and cryopreservation procedures in dogs and cats. Furthermore, information obtained by CASA systems could also be important when monitoring for example the effect of environmental stress on spermatozoa and for toxicity studies. In cats, CASA is less documented, and most studies describe the characteristics of epididymal sperm, which is frequently used for in vitro fertilization in cats. Implementation of the CASA technique in cat reproduction could be interesting to further optimize assisted reproductive techniques in domestic cats and endangered wild felids.

Use of fluorescent stainings and flow cytometry for canine semen assessment.

Over the last few decades, appreciable progress has been noted in canine semen assessment techniques. The common use of accurate and sensitive diagnostic methods, such as computer-assisted sperm analysis (CASA), flow cytometry and sperm penetration tests have become routine procedures in specialized andrology laboratories. Many fluorescent probes have been applied to the assessment of specific sperm characteristics in dogs. Flow cytometry enables the observation of cell characteristics such as size, shape and function of the spermatozoon, that can be revealed by a fluorochrome or fluorescent label. The analysis of events detected on dot plots gives accurate and highly reliable information on membrane integrity, acrosomal status, mitochondrial activity, capacitation status, lipid peroxidation, apoptosis and DNA damage. Despite the development of these modern and accurate tools, it is still questionable if the ideal method of semen evaluation, allowing predicting of the fertilizing potential of semen, has been established.

Effect of dilution temperature on boar semen quality.

As boar semen is very sensitive to cold shock and changes in temperature during semen processing can have a profound impact on semen quality, the effect of the extender temperature at the time of dilution was investigated in a two-step dilution protocol for boar semen being processed for liquid storage. Fifteen boars of different breeds and ages from a commercial artificial insemination centre were included. One ejaculate per boar was collected and processed with Beltsville Thawing Solution semen extender. Each ejaculate was diluted (1 : 1) at 30 °C, and subsequently, the samples were diluted (30 × 10(6) sperm/ml) with either preheated extender [29.3 °C ± 0.2 °C, group A (GA)] or extender at room temperature [22.7 °C ± 0.6 °C, group B (GB)]. Samples were transported to the Faculty of Veterinary Medicine (University of Ghent, Belgium) in two isotherm boxes (one per group), stored at 17 °C and investigated for three consecutive days (D0 to D2). At D0, D1 and D2, motility parameters [computer-assisted semen analysis (CASA)] and the per cent of sperm with intact membrane (% IM) by eosin nigrosin staining were evaluated. At D0 and D2, the % of sperm with intact acrosome (% IA) was studied by Pisum sativum agglutinin staining. The average temperature of the 1 : 1 dilution was 29.4 °C ± 1.1 °C immediately after extender addition. No significant differences were found between groups for per cent motility [79.3 ± 9.0 for GA and 81.1 ± 9.2 for GB (p = 0.372)], % progressive motility [56.5 ± 13.3 for GA and 58.4 ± 13.8 for GB (p = 0.737)] or any CASA parameter. No differences were found for % IM [85.1 ± 10.7 and 84.5 ± 3.8 for GA and GB, respectively (p = 0.761)] and % IA [72.2 ± 9.4 for GA and 68.3 ± 16.6 for GB (p = 0.792)]. In conclusion, when a two-step dilution is performed, preheating the extender for the second dilution to match the semen temperature did not result in better semen quality compared to a dilution at a moderate room temperature.

Influence of sampling factors on canine sperm motility parameters measured by the Sperm Class Analyzer.

The aim of the present study was to investigate the effect of different technical settings and semen processing on sperm motility parameters measured by the Sperm Class Analyzer (SCA). Semen was collected from 3 dogs, pooled, and diluted in phosphate buffered saline and subsequently assessed by the SCA for the different sperm motility characteristics. The data were statistically analyzed by ANOVA and the repeatability was assessed by coefficient of variation (CV). After a principal component analysis, the reliability was determined with intra-class correlation coefficient (ICC). In experiment 1, the CV's were below 10% for all evaluated parameters. Significant differences (P < 0.05) were found between the different sperm concentrations (25, 50, and 75 x 10(6) spermatozoa/ml) in all of the motion parameters assessed, yielding the highest ICC (0.81) at 25 x 10(6) spermatozoa/ml. No significant differences (P > 0.05) in SCA read-outs were found between the number of microscopic fields captured (1, 2, 3, 4, or 5 fields), yielding the highest ICC (0.83) when 3 fields were captured. No significant differences (P > 0.05) in motility parameters were found between the number of cells analyzed in each field (20, 50, and 100 spermatozoa) with the exception of beat cross frequency. Reliability of the SCA was good (ICC = 0.71 to 0.90) for all motility measurements when 20 (ICC = 0.89) or 50 (ICC = 0.77) cells were captured in each field, but only just acceptable (ICC = 0.51 to 0.70) when 100 cells were counted (ICC = 0.67). The frame settings significantly (P < 0.05) influenced most of the measured motility characteristics. Scanning 60 frames at a frame rate of 30 Hz improved the reliability of the results (ICC = 0.92). In conclusion, we suggest that the measurements with the SCA are ideally performed at a sperm concentration of 25 x 10(6) spermatozoa/ml, counting at least 100 cells in three microscopic fields. We also propose that the SCA should analyze 60 frames at 30 Hz to yield consistent results of a set of measurements or a measuring instrument thus obtaining reliable motility results.

In vitro evaluation of fresh sperm quality in tomcats: a comparison of two collection techniques.

The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P<0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P<0.01), while CT sperm contained more spermatozoa with tail abnormalities (P<0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P>0.05) between CT and EP sperm. Nevertheless, no difference (P>0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa.

DNA fragmentation in canine immature grade 1 cumulus-oocyte complexes.

In this work, we studied the incidence of DNA fragmentation, interpreted as apoptotic changes and assessed by the TUNEL assay, in cumulus cells and oocytes of immature Grade 1 cumulus-oocyte complexes (COCs) obtained from healthy bitches (n = 27) of three age groups: young (1-3 years; n = 13), adult (4-6 years; n = 8) and elderly (7-10 years; n = 6). Age affected (p < 0.05) Grade 1 COCs recovery rates, with young animals yielding more (p < 0.01) Grade 1 COCs than the other two age groups. Conversely, no differences were observed in the incidence of DNA fragmentation (TUNEL-positive) in cumulus cells or oocytes between the three age groups. Overall, more than 80% of Grade 1 COCs presented <15% of TUNEL-positive cumulus cells and enclosed TUNEL-negative (intact DNA) oocytes. Despite a higher proportion of TUNEL-negative oocytes being found in the germinal vesicle stage, most of the oocytes with nuclear material compatible with meiosis resumption (MR) or with non-identifiable nuclear material (ND) did not present DNA fragmentation. No correlation was observed between DNA fragmentations in oocytes and in cumulus cells. We concluded that the morphological parameters used to classify canine Grade 1 COCs are reliable to select a homogeneous population of COCs with low incidence of DNA fragmentation. Furthermore, these results indicate that DNA fragmentation can only explain a minor proportion of the incidence of MR and degeneration in canine oocytes at collection.

Effect of organic selenium in the diet on sperm quality of boars.

The effect of a diet supplemented with organic selenium (Se) on sperm production and quality of boars was investigated. Sixty mature boars from a commercial artificial insemination centre were randomly allocated at Day (D) 0 into Group A and B. Group A received the regular ration supplemented with inorganic Se (0.4 mg/kg feed as Na(2) SeO(3)) whereas Group B was switched to the same diet but with organic Se (0.4 mg/kg fed as Se-yeast). The sperm was investigated during 4 months (D0, D30, D60, D75, D90, D105 and D120). Sperm concentration and motility were objectively measured using a photometer and Computer Assisted Semen Analysis (CASA) respectively. Morphology of the sperm was assessed using eosin-nigrosin staining and the resistance to induction of oxidative stress (production of malonaldehyde, MDA) through thiobarbituric acid reagent species analysis. Additionally, the Se concentration in sperm and blood plasma were measured. Repeated measures analysis of variance (anova) from D60 to 120 (spermatogenesis of approximately 2 months) or anova at D120 (Se concentrations) were used for statistical analysis. The total number of ejaculated sperm was not significantly different between both groups, but boars of Group B had a significantly higher sperm concentration (434.6 vs 514.1 × 10(6) sperm/ml; p < 0.05). Small differences (p < 0.05) were observed between both groups for some CASA parameters, namely straight line velocity (µm/s) (Group A: 48.3, Group B: 45.1), straightness (%) (Group A: 65.6, Group B: 62.2) and linearity (%) (Group A: 32.2, Group B: 29.3). The sperm of Group B showed more oxidative stress (4.1 vs 4.9 µmol MDA/l; p < 0.05) compared with those of Group A. No significant differences (p > 0.05) were observed for the other parameters. Under the present study conditions, changing from inorganic Se to organic Se in the diet of boars increased sperm concentration but reduced some motility parameters and resistance to oxidative stress.

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 degrees C) on sperm quality.

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR((R))14-PI) and DNA fragmentation (TUNEL). After addition of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 degrees C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P<0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.

Diseases in swine transmitted by artificial insemination: an overview.

Artificial insemination (AI) of swine is widely practiced in countries with an intensive pig production. It is a very useful tool to introduce superior genes into sow herds, with minimal risk for disease transmission. However, the impact of semen that is contaminated with pathogens can be enormous. Most of the micro-organisms that have been detected in boar semen are considered non-pathogenic, but some are known pathogens (e.g. porcine reproductive and respiratory syndrome virus) that can cause major economic losses. Microbial contamination of semen can be due to systemic and/or urogenital tract infections of the boar, or can occur during collection, processing and storage. It can result in reduced semen quality, embryonic or fetal death, endometritis and systemic infection and/or disease in the recipient female. Conventional techniques for isolation of bacteria and viruses from the semen do not always provide optimal results for various reasons, including lack of sensitivity and speed of testing, and difficult interpretation of the outcome. More recently, PCR tests are commonly used; they have a high sensitivity, the outcome is quickly obtained, and they are suitable for monitoring a large number of samples. The best strategy to prevent AI-transmitted diseases is to use boars that are free of specific pathogens, to monitor the animals and semen regularly, and to maintain very high biosecurity. Additional measures should be directed at treating semen with appropriate antimicrobials, and at reducing contamination during semen collection, processing, and storage.

Effect of body weight, age and breeding history on canine sperm quality parameters measured by the Hamilton-Thorne analyser.

During the last decade, several computer assisted sperm analysis (CASA) systems have been validated for canine sperm quality assessment. Regarding the impressive possibilities of these systems, further research is required to determine which CASA measurements are of clinical importance in canine andrology. In the present study, the sperm quality parameters obtained by the Hamilton-Thorne Semen Analyser (Ceros 12.1; HTR) were correlated with the body weight and the age of the dogs. Moreover, the sperm quality parameters of dogs with a different breeding history were compared. The sperm-rich fraction was collected from 111 dogs of 50 different breeds, which were presented at our department. Immediately after collection, the concentration, the total sperm output (TSO) and 13 different sperm motility and velocity characteristics were measured by the HTR. The percentage of live spermatozoa and the spermatozoal morphology were examined on eosin/nigrosin stained smears. Based on their breeding history, the dogs were divided in three groups: 'fertile' (n = 60), 'subfertile' (n = 17) or 'not used for breeding' (n = 34). Significant (p < 0.05) correlations were established between the body weight of the dogs and the TSO (r = 0.245) and velocity curvilinear (VCL; r = -0.220), respectively. The age was negatively correlated with the percentage of normal spermatozoa (r = -0.203; p < 0.05). The correlations with all the other evaluated sperm parameters were low and not significant. Significant differences between the 'fertile' and the 'subfertile' group were found for all of the evaluated sperm quality parameters (except for BCF, LIN, STR and MEDIUM). In conclusion, dogs tend to produce ejaculates with a lower percentage of normal spermatozoa with increasing age and dogs with higher body weights produce ejaculates with a higher TSO and a lower VCL. Significantly poorer sperm characteristics were found for dogs with lower in vivo fertility results.

Comparison of computer-assisted sperm motility analysis parameters in semen from Belgian blue and Holstein-Friesian bulls.

Subjective microscopic sperm motility results have recently been demonstrated to differ between Holstein-Friesian (HF) and Belgian Blue (BB) bulls. However, such assessments are rather imprecise. In the present study, sperm motility was assessed objectively by means of the Hamilton Thorne CEROS version 12.2c computer-assisted sperm motility analyser (CASA), and differences between the BB and HF breed could also be demonstrated. Higher percentages of both totally (p < 0.0001) and progressively (p < 0.0001) motile spermatozoa were encountered in the HF breed compared with the BB breed. Furthermore, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower beat cross-frequency (p = 0.0007) combined with a higher lateral head displacement (p = 0.0015), was the basis for the lower velocity of BB sperm cells. Additionally, BB spermatozoa move less straight forward, resulting in a lower straightness (p < 0.0001). No sperm motility differences were observed between age groups within the BB breed. The breed differences were observed in the examined bull populations residing at AI centres, in Belgium for the BB bulls and in the Netherlands for the HF bulls. However, these bull populations are selected for fertility. A similar pattern was observed in an unselected bull population of both breeds, although these differences were mostly non-significant for the different CASA parameters. Nevertheless, these data suggest that a genetic component might be responsible for the observed sperm motility breed differences.

Air contact influences the pH of extended porcine semen.

In this study, the pH-rise during storage of extended porcine semen was examined. This pH-rise was found to be caused by CO(2)-loss from the buffering system in the extender and was more pronounced with increasing air volume in the recipient. An influence on sperm motility parameters was observed between semen samples stored in the presence of different amounts of ambient air in the recipient. Velocity parameters and percentage motile spermatozoa were significantly lower for semen stored in recipients with higher air volume and elevated pH. Adjusting extender preparation by avoiding air contact in commercial AI-centres may minimize the pH-rise and its influence on sperm motility.

True hermaphroditism in six female littermates after administration of synthetic androgens to a pregnant bitch.

A pregnant bitch was treated with a synthetic testosterone mixture on approximately day 40. The female offspring (six pups) showed an increased anogenital distance, vaginal enlargement and a variable amount of vaginal discharge. The urinary orifice was found dorsally in the vestibulum, stooled on a protruding phallus-like structure. All six pups underwent a laparotomy and subsequent spaying and a modified ventral episioplasty technique to lift up the labia to a more vertical position in order to prevent urine accumulation. Histopathological examination of the genital tracts demonstrated the presence of bilateral ovotestis and remnants of the Wolffian duct system in all cases. The finding of true hermaphroditism of the offspring after exogenous androgen administration during gestation of the bitch has not yet been reported elsewhere.

New techniques for the assessment of canine semen quality: a review.

Until recently, canine semen assessment was routinely performed by conventional light microscopic techniques. The limitations of these methods include subjectivity, variability, the small number of spermatozoa analyzed, and poor correlation with fertilizing potential. The last decade, several new in vitro techniques have been introduced for canine semen assessment that enable a more detailed evaluation of several sperm characteristics. Numerous fluorescent staining techniques have been developed for the evaluation of specific sperm characteristics and functions, including plasma membrane integrity, capacitation status and the acrosome reaction. By combining fluorescent stains, several functional sperm characteristics can be assessed simultaneously. Moreover, by means of flow cytometry, large numbers of fluorescently labelled spermatozoa can be analysed in a short interval. Following thorough standardization and validation, computer-assisted sperm analysis systems provide objective and detailed information on various motility characteristics and morphometric dimensions that cannot be identified by conventional light microscopic semen analysis. In vitro assays, evaluating the capacity of canine spermatozoa to bind to the zona pellucida or oviductal explants, or to penetrate the oocyte, provide additional information on canine gamete interaction that may be useful in predicting the fertilizing potential of spermatozoa. Although substantial improvements have been made in canine semen assessment, surprisingly few parameters were correlated with in vivo fertility. Therefore, further research is required to determine which sperm characteristics are of clinical value for predicting the in vivo fertility in dogs.

Validation and usefulness of the Sperm Quality Analyzer (SQA II-C) for bull semen analysis.

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