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

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

The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen

Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R²=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R²=0.84, P<0.05) was observed between real and predicted fertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates.     

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility.     

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.     

Variability in relationships between semen quality and estimates of in vivo and in vitro fertility in boars

The present experiment was designed to characterize relationships between common semen quality and fertility estimates for three boars known to differ in farrowing rate, number of pigs born alive, and monospermic penetration rate. The approach chosen to accomplish this was to monitor semen quality from these boars and use their semen alternately for either artificial insemination or in vitro fertilization for 40 weeks. This strategy relied on the variability in semen quality parameters that normally occurs in an individual boar over time. When comparisons were made among boars, farrowing rates, numbers of pigs born alive, and monospermic penetration rates were significantly different, but progressive motility, normal head and tail morphology, and acrosome morphology were not. However, when comparisons were made among ejaculates within individual boars, there were significant effects of semen quality on both in vivo and in vitro fertility. For boar 3495, the proportion of spermatozoa exhibiting progressive motility and distribution of spermatozoa in a percoll gradient had a positive linear effect on number born alive and monospermic penetration rate, respectively. For boar 2901, quadratic equations best described changes in litter size as a function of progressive motility and normal acrosomes. In addition, monospermic penetration rate increased linearly as normal acrosomes and the proportion of spermatozoa recovered from a percoll gradient increased. For boar 4291, the relationship between progressive motility and number born alive and between normal acrosomes and number of pigs born alive were also quadratic. However, a significant linear relationship was present only between normal acrosomes and monospermic penetration rate. These results demonstrate that simply relying on the means of common semen quality estimates from some boars has limited value in terms of being used as a prospective indicator of their in vivo or in vitro fertility. In contrast, characterization of relationships between semen quality and fertility estimates is useful for estimating differences in the fertility of ejaculates from individual boars. However, both quantitative and qualitative differences in these relationships among boars are present and a given semen quality estimate that is a good predictor of in vivo or in vitro fertilization for one boar, may not be applicable for others.     

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

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

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation.     

Motility characteristics of boar spermatozoa after addition of prostaglandin F2alpha

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

Fertility prediction in dairy goats from Murciano-Granadina breed: The role of sperm evaluation and female traits

Fertility is one of the most economically important traits in farm animals, due to the direct and indirect costs associated to low pregnancy rates. Thus, one of the priority goals in animal reproduction is to predict the performance that the semen doses will have in vivo based on the quality values obtained in laboratory assays. Attempts have been made for getting a predictive model of fertility of frozen-thawed sperm in dairy goats, but similar studies have not been conducted for chilled goat buck sperm doses that are mostly used for artificial insemination in many countries including Spain. We study how parameters of in vitro sperm quality and characteristics of Murciano-Granadina dairy goats may affect the in vivo fertility obtained after artificial insemination with semen doses chilled at 4 °C. Moreover, this information was used for obtaining predictive models of the fertility. Sixty-three ejaculates from 13 males were used to prepare chilled doses for the insemination of 495 goats over 13 sessions. Fresh and chilled sperm were evaluated for motility and plasma membrane integrity with a computer-assisted sperm analysis system and flow cytometry, respectively. Fertility was determined at parturition, according to the kidding goats. Overall fertility was 59.6%. Pearson’s correlation coefficients between in vivo fertility and quality variables of fresh sperm were not significant and were low (below 0.34 in absolute value) for chilled sperm. Females’ characteristics had a low negative impact on fertility (correlation coefficients of ?0.19 with age, ?0.20 with parturitions and ?0.11 with total milk yield obtained in the best lactation). Fixed and mixed logistic regression procedures were used trying to explain the fertility results. None of the models accurately predicted fertility, but the best models included the percentage of total motile sperm or average path velocity from fresh semen, age of the females and the session effect (uncontrolled environmental effects). These analyses showed that primiparous goats were 2.42 times more likely to get pregnant than goats that had kidded four or more times. Our field assay data on fertility in Murciano-Granadina dairy goats highlighted the importance of making quality controls of sperm, of choosing the doses presenting high percentages of motile sperm exhibiting regular trajectories and of selecting the youngest goats for AI, after their first kidding. Efforts should continue to obtain better predictive models for improving fertility in goat dairy herds.     

Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis

The fertility of liquid-preserved boar semen declines during storage at 17°C, insemination trials even indicating early losses in fertilizing ability within the first 24-48 h of storage. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 17°C and stored for 12, 24, 72, 120, and 168 h before investigation. At each time, basic semen quality was characterized by sperm motility and viability. Subsequently, washed subsamples were incubated in variants of an in vitro fertilization (IVF) medium and assessed for kinetic changes of viability (plasma membrane integrity) and intracellular calcium concentration using flow cytometry in combination with propidium iodide and Fluo-3. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. During storage, standard semen parameters remained on a high level. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. Assessment of response to capacitating media by flow cytometry appears a markedly more sensitive way of monitoring sperm functionality during storage than the standard semen parameters of motility and viability.     

Motility and fertility of alginate encapsulated boar spermatozoa

Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 degrees C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16-18 degrees C, and then were stored at 5 degrees C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 degrees C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 degrees C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8h incubation at 37 degrees C after storing for over three days (P<0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability.     

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

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

The predictive value of routine semen evaluation and IVF technology for determining relative boar fertility

Practical techniques for assessing semen quality in order to predict male fertility are still needed. The principal objective of this experiment was to evaluate routine laboratory evaluation and in vitro fertilization (IVF) techniques as predictors of relative boar fertility using a low-dose AI protocol. Nine boars were evaluated during a 6.5+/-1 mo period, beginning at 29-32 wk of age. Ejaculates were evaluated for motility, morphology and concentration, diluted to 1.5 billion sperm in 50 mL extender, and used to breed 50+/-5 gilts over the same period. On nine occasions, a specific aliquot of the ejaculate's first sperm-rich fraction was evaluated using IVF procedures. Boars differed (P<0.001) consistently for pregnancy rate (from 73 to 98%), farrowing rate (71-98%) and total born (8.8-12.0). Routine semen evaluation and IVF parameters that presented significant differences between boars, but no differences in time and no boar by time interaction, were used to correlate in vivo fertility. A multiple regression model based on routine semen evaluation parameters accounted for up to 27 and 22% of the variation of fertility index and total piglets born, respectively, whereas male pronuclear formation rate was the IVF variable that accounted for 17 and 12% of the variation in farrowing rate and fertility index, respectively. Collectively, we inferred that the use of low sperm numbers for AI, determination of pregnancy rate at Day 30, motility of extended semen after 7 and 10d, and specific IVF parameters may be useful for identifying relatively infertile boars that are not currently excluded from use in existing commercial boar studs.     

The value of microscopic semen motility assessment at collection for a commercial artificial insemination center, a retrospective study on factors explaining variation in pig fertility

This study was conducted to evaluate the relationship between boar and semen related parameters and the variation in field fertility results. In 8 years time semen insemination doses from 110 186 ejaculates of 7429 boars were merged to fertility parameters of inseminations of 165 000 sows and these records were used for analysis. From all ejaculates boar and semen related data were recorded at the artificial insemination (AI) centers. Fertility parameters, such as farrowing rate (FR), ranging between 80.0% and 84.0%, and the total number of piglets born (TNB), ranging between 12.7 and 13.1, were recorded and from these the least square means per ejaculate were calculated. Only 5.9% of the total variation in FR was due to boar and semen variability of which 21% (P = 0.0001) was explained by genetic line of the boar, 11% (P = 0.047) was explained by laboratory technician, and 7% (P = 0.037) was explained by the AI center. For TNB the total variation was 6.6% boar and semen related of which 28% (P < 0.0001) was explained by genetic line of the boar and 7% (P = 0.011) was explained by the AI center. Only 4% of the boar and semen related variation was caused by sperm motility (microscopically assessed at collection, ranging from 60% to 90%). Other variation in FR and TNB was explained by management and semen related parameters (age of boar, 3%; P = 0.009; and 8%; P = 0.031, respectively), days between ejaculations (1%; P < 0.0001 of FR), number of cells in ejaculate (1%; P = 0.042 of TNB), year (9%; P = 0.032), and 13%; P = 0.0001, respectively), and month (11%; P = 0.0001; and 5%; P = 0.0001, respectively). Although semen motility is considered an important parameter to validate the quality of the ejaculate processed, it only minimally relates to fertility results under the current Dutch AI practice. Other boar and semen related parameters, like genetic line of the boar, are more relevant factors to select boars for AI purposes.     

Can external quality control improve pig AI efficiency?

External quality control programmes carried out by central laboratories have been long established in human andrology with the aim of enhancing the accuracy and reproducibility of semen assessment. Compared to human, demands on boar semen assessment in AI stations are more complex, with the need both to identify boars with poor ejaculate quality and to monitor individual boar differences for semen storage. Additionally, appropriate assessment serves as a control instrument to ensure the security and efficiency of semen processing. Despite current limitations regarding the ability of sperm assays to estimate the potential fertility of males, it is evident that boar fertility is related to certain conventional semen tests, e.g. sperm morphology. In central studies carried out on stored semen from 11 AI stations, flow cytometric assessment of plasma and acrosome membrane integrity proved to be more sensitive in detecting sperm damage associated with ageing and temperature stress as compared to light microscopy. Membrane integrity of stored semen differed between AI stations indicating significant influences of semen processing on sperm quality. Thus external control of semen quality in reference laboratories may be useful to monitor the efficiency of internal semen quality control in individual AI stations, to identify males with lower semen quality and/or poor response to semen storage, and to verify the precision of sperm counting. The possibility that central laboratories with sufficient resources may be able to identify functionally different responding sperm subpopulations for better estimation of fertility is discussed. Ideally, external quality control schemes for AI stations would comprise application of validated tests with high relevance for fertility (including bacterial status), analysis of semen processing on the AI station, and training courses for laboratory personnel.     

Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen

Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests.     

Perspectives de l’analyse de la mobilité des spermatozoïdes

Several studies have shown a good correlation between sperm motility and fertility though the microscopic evaluation of the percentage of motile sperm is highly subjective by nature. Therefore in the last decade, various objectives methods have been proposed to overcome this problem. Two types of methods were developed: The methods based on the analysis of images obtained by microphotography, microcinematography and microvideography and the global, undirect methods based on physical principles. Several systems based on video and image analysis (Computer Aided Sperm Analysis, CASA) have been developed and are used in numerous laboratories of reproductive biology. CASA technology offers the possibility to analyse some characteristics of sperm motion which are related to the fertilization potential and to develop new parameters related to some important aspects of sperm behavior such as hyperactivation. However, there is a large amount of interactions between the operator and the CASA machine. CASA instruments are not “ready-to-use” robots: the reliability of CASA depends largely on the expertise and training of the user and the application of standardized procedures and quality control schemes. By contrast, there is only minimal interaction between the operator and the Sperm Quality Anlyser which is a new device measuring and index of sperm motility highly correlated to the concentration of progressively motile sperm. The device uses light passed through a small sample of semen introduced in a capillary tube to detect variations in optical density that result from moving particles. The reproducibility of the measurements is excellent, the device is easy to use and this is a potentially useful tool for field-work studies. Further investigations of this device in the managment of male infertility is warranted. Finally, both types of objectives approaches are complementary to the conventional analysis of sperm motility and they will not replace it. Standardized procedures have been proposed by the World Health Organization for the subjective evaluation of sperm motility. Such procedures are very useful to reduce significantly the intra- and interlaboratory variations but internal and external quality controls schemes indicate that they are not sufficient to achieve acceptable levels of variation and regular quality controls followed by the definition and the application of corrective procedures are required.     

Predictors of success of semen cryopreservation in chickens

Semen cryopreservation is very important for the ex situ management of genetic diversity in birds but it is rarely used. This is partly because of the highly variable success rates, and this emphasizes the need for predictors of semen freezability. This study evaluated the ability of semen quality tests to predict the success rates of semen cryopreservation in chickens and the relationships between each test. Individual variations of in vitro quality tests of semen were compared to the fertility obtained with fresh and cryopreserved semen. The in vitro semen quality tests represented viability, integrity, motility (percentage of viable and morphologically normal cells (PVN); mass motility (MMOT) and different motion parameters including percentage of motile spermatozoa (PMOT)) and biophysical tests (OSM, resistance to osmotic stress; membrane fluidity (FLUID)). Different in vitro tests were significantly correlated between each other for fresh (MMOT, PVN and FLUID, many criteria of objective motility) and cryopreserved semen (MMOT, different objective motility parameters, PVN). Fertility was significantly correlated with PVN for fresh semen and PVN and different objective motility criteria for cryopreserved semen. Membrane fluidity, followed by PVN, PMOT and MMOT, measured on fresh semen samples was positively correlated with fertility obtained with cryopreserved semen. The combination of the first three tests explained 85% of the variability of fertility observed with cryopreserved semen. In conclusion, we showed that different in vitro tests of semen quality are of predictive value for the success rate of semen cryopreservation in the chicken, the most accurate being membrane fluidity.