Retrospective Analysis of Canine Semen Evaluations with Special Emphasis on the use of the Hypoosmotic Swelling (HOS) Test and Acrosomal Evaluation Using Spermac®

Routine semen evaluation includes volume, motility, vital staining for live‐dead ratio and pathomorphology including Spermac^® staining for evaluation of the acrosome. In recent years, depending on the species, also the hypoosmotic swelling (HOS) test has been applied routinely for evaluation of semen quality. In this respect, a significant correlation between the ability of spermatozoa to swell in HOS test and the fertilizing ability has been reported. Also for evaluation of dog semen, reference has been made to the HOS test; however, its correlation to conventional semen parameters so far is discussed controversially. In the present study, the results of 400 semen examinations from stud dogs presented at our clinic were evaluated for their correlations between conventional semen parameters (motility, live/dead ratio, pathomorphology), conventional semen parameters and age, Spermac^® staining and HOS test, respectively. We found a significant correlation of age and sperm concentration (p < 0.01), total sperm count (p < 0.0001), percentage of progressively motile sperm (p < 0.01) and live spermatozoa (p = 0.012). Furthermore, several correlations between conventional semen parameters were identified. Percentage of sperm with normal acrosome identified by Spermac ^® staining correlated significantly with live spermatozoa (p < 0.0001) and percentage of progressively motile sperm (p < 0.01). A significant correlation was proven between curled tails in HOS test and age (p < 0.001), motility (p < 0.0001), live sperm (p < 0.0001), acrosomal status (p < 0.05), pathomorphology (p < 0.0001) and sperm concentration (p = 0.011). These results indicate that Spermac^® staining and the HOS test are useful in improving canine semen analysis.     

Evaluation of membrane integrity of canine epididymal spermatozoa by short hypoosmotic swelling test with ultrapure water

The effectiveness of the water test, short hypoosmotic swelling test with ultrapure water was examined in canine epididymal spermatozoa to evaluate tail membrane integrity. Spermatozoa during epididymal transit were also characterized. Sperm suspension obtained from cauda epididymis was diluted 1:4 with ultrapure water, and incubated for 5 min. The percentage of swollen spermatozoa in the water test was significantly correlated with both the sperm motility and the swelling value obtained by the conventional hypoosmotic swelling test. Canine spermatozoa collected from the caput epididymis were not motile, but revealed membrane integrity in a water test. The water test can be used as a simple and short hypoosmotic swelling test to evaluate the tail membrane integrity of canine epididymal spermatozoa.     

Assessment of bovine X- and Y-bearing spermatozoa in fractions by discontinuous percoll gradients with rapid fluorescence in situ hybridization

This study was designed to apply the method of discontinuous Percoll gradients for sex preselection in bovine semen by using a current developed molecular technique, fluorescence in situ hybridization (FISH). In addition, we attempted to amplify the level of enrichment of X- or Y-bearing spermatozoa by treating for activating sperm motility performance with 10 mM caffeine. Bovine spermatozoa were fractionated on Percoll gradients into two major subpopulations of motile spermatozoa (bottom fraction) and weak motile spermatozoa (top fraction). The percentage of Y-bearing spermatozoa in the top fraction (52.9%) slightly exceeded and that in the bottom fraction (44.3%) decreased significantly (P<0.001) compared with the theoretical ratio (50:50). Washing sperm with BO medium affected a deviation between the two sex populations, whereas semen activated with caffeine showed no difference in the percentage of X- and Y-bearing spermatozoa in both fractions compared with the theoretical ratio (50:50). These results show that the proportion of X- and Y-bearing bovine spermatozoa can deviate after discontinuous Percoll gradients, although the proportion of X- and Y-bearing bovine spermatozoa was affected by sperm motility of the sample applied.     

Relationship Between Motility and Membrane Integrity of Boar Spermatozoa in Media Varying in Osmolality

A study was conducted to evaluate the relationship between boar sperm motility and membrane integrity following exposure to media with 150–1120 mOsm. Total sperm motility was defined as the percentage of spermatozoa that had any form of motility was subjectively assessed under a light microscope. Sperm cell damage was expressed as a loss of membrane integrity as measured by a combination of fluorescent stains, carboxyfluorescein diacetate (CFDA) and propidium iodide (PI), and Hoechst 33258 (H33258). There were no significant differences between sperm motility and membrane-intact spermatozoa, as measured by CFDA-PI and H33258, in media with 250 and 300 mOsm. In anisosmotic conditions, a higher amount of membrane-intact spermatozoa than motile spermatozoa was observed. In hypo-osmotic conditions (150 mOsm), a high proportion of spermatozoa had curled or coiled tails and most of them retained their entire membrane integrity, as detected by CFDA-PI. In media with 350–1120 mOsm, some spermatozoa accumulated PI in the head region and CFDA in the mid-piece. These spermatozoa fluoresced blue at the lower region of the head, as detected by H33258. The ATP content in spermatozoa exposed to hypo- and hyperosmotic conditions was markedly reduced. There was no recovery of sperm motility on returning the spermatozoa to isosmotic conditions after 10 min incubation in anisosmotic conditions, indicating that the spermatozoa suffered an almost complete and irreversible loss of motility. This irreversible loss of motility may be a consequence of reduced ATP production in spermatozoa subjected to anisosmotic conditions. The results of this study demonstrate that plasma membrane integrity assessment in combination with sperm motility, using a range of media varying in osmolality, can give valuable information about the status and function of different sperm membranes, which might be relevant for semen preservation.     

Utilisation of a sperm quality analyser to evaluate sperm quantity and quality of turkey breeders

1. A relatively new instrument known as a Sperm Quality Analyzer (SQA) offers a rapid assessment of sperm quality and quantity by providing a sperm quality index (SQI). The SQA measures a combination of the intensity of sperm activity and motile concentration by determining the number and amplitude of sperm movements per second in a capillary tube as detected through light beam interference. 2. Because the SQA has not been tested for its potential use in turkeys, the objective was to determine if the SQA could accurately respond to changes in turkey sperm concentration, viability, and motility in semen collected from turkey breeders. 3. The effect of varying concentrations of sperm on SQI values was evaluated by diluting replicate pools of semen from 4 different aged turkey breeder flocks with saline. Results from all 4 flocks showed that semen dilutions greater than 20-fold resulted in a linear decline in SQI values. 4. Additional in vitro analysis evaluated the effects of turkey sperm viability on the SQI under conditions of constant sperm concentration. Incubated, live sperm was mixed in various proportions with thawed, dead sperm to determine changes in viability. Increased proportions of dead sperm caused a decline in the SQI. 5. To assess sperm motility, turkey semen was incubated under either aerobic (motile) or anaerobic (immotile) conditions. Varied amounts of immotile and motile sperm samples were mixed. A linear increase in the SQI was observed as per cent motile sperm increased. 6. These results indicate that the SQA can respond to differences in turkey sperm concentration, viability, and motility using in vitro analyses.     

Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid‐reactive species (TBARS) assay and the 2, 4‐dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.     

Effects of bovine serum albumin and trehalose in semen diluents for improvement of frozen-thawed ram spermatozoa

In this study, two following experiments were performed to improve post-thaw motility and viability of frozen-thawed ram spermatozoa. We examined i) the effects of different concentrations of bovine serum albumin (0, 0.3, 1, 5, 10 and 15% BSA) in semen diluents lacking egg yolk and ii) the effects of four semen diluents, fructose (F: control) and trehalose (T) in semen diluents containing egg yolk, 15% BSA in semen diluents without egg yolk (BSA), and modified phosphate buffered saline (m-PBS). Frozen-thawed spermatozoa were examined for progressive sperm motility, viability, morphological abnormality, sperm tail swelling test, and sperm acrosome integrity. In Experiment 1, the rates of sperm motility immediately after thawing (0 h) were significantly (P<0.05) higher in the 10 and 15% BSA groups (55.0 +/- 2.9 and 58.3 +/- 6.7%, respectively) than in the positive control (F) group (41.7 +/- 4.4%). The rate of sperm viability in the negative control (0% BSA) group (80.2 +/- 3.3%) was significantly (P<0.05) lower than in the positive control (F) group (89.8 +/- 1.5%), but when compared with the F group, no significant differences were found among the 0.3, 1, 5, 10 and 15% BSA groups at 0 h. The rates of sperm morphological abnormality of the 10 and 15% BSA groups (6.5 +/- 1.3 and 6.3 +/- 1.1%, respectively) were significantly (P<0.05) lower at 0 h than that in the 1% BSA group (16.3 +/- 5.2%). In Experiment 2, T addition improved (P<0.05) the post-thaw motility compared with the F and BSA groups. Furthermore, at 3 and 6 h, the post-thaw motility of the T group (36.3 +/- 2.4 and 25.0 +/- 2.0%, respectively) was significantly (P<0.05) higher than in the BSA (26.3 +/- 2.4 and 18.8 +/- 1.3%, respectively) and F (28.8 +/- 3.8 and 18.8 +/- 2.4%, respectively) groups. The post-thaw sperm motility and viability in the m-PBS group were significantly (P<0.05) lower than those of the control (F), T, and BSA groups throughout all observation points. These results indicate that 10 and 15% BSA can be substituted for egg-yolk for ram semen diluent and that the addition of trehalose enhances motility and viability of ram spermatozoa after freezing and thawing.     

Use of the Sperm Quality Analyzer (SQA II‐C) for the Assessment of Dog Sperm Quality

In the present study, an automated system for sperm analysis, the Sperm Quality Analyzer (SQA II‐C), was tested as a potential tool for the assessment of dog sperm quality. In the first experiment the device displayed a good repeatability of measurements for semen of medium and high quality, as evidenced by a low coefficient of variance (CV; 0.08), whereas a high CV (0.46) was obtained for one dog with semen of inferior quality. In the second experiment, seven different sperm concentrations (25–300 ×10⁶/ml), obtained by dilutions in Hepes‐TALP medium were stored for 48 h at room temperature. A concentration dependent increase in sperm motility index (SMI) was shown, reaching a plateau at 150×10⁶ spermatozoa/ml. For all sperm concentrations, the SMI value decreased significantly after 24 h, indicating the importance of sperm motility for SMI values. For sperm concentrations lower than 150×10⁶/ml, highly significant correlations [r=0.80;p<0.05] were established between SMI values on one hand and sperm concentration, and semen parameters expressing the overall semen sample quality on the other hand (experiment 3) while non‐significant or low correlations were found between SMI values and other individual sperm parameters. In experiment 4, significantly high correlations (r=0.97) were found between mean SMI values and post‐thaw motility and progressive motility assessed subjectively. In conclusion, our study indicates that both motility and concentration largely influence SMI values and that the SQA II‐C saturates at 150×10⁶ fresh spermatozoa/ml. In our opinion, the SQA II‐C may be a useful and objective device to assess the post‐thaw motility of dog sperm.     

Effect of Sperm Cryopreservation on the European Eel Sperm Viability and Spermatozoa Morphology

The main objective of the present work was to study the effect of cryopreservation of European eel sperm both on the sperm viability and the spermatozoa head morphology. Spermatozoa morphology was evaluated with computer-assisted morphology analysis after collection in fresh samples, after adding the freezing medium containing dimethyl sulfoxide as cryoprotectant and, finally, after the cryopreservation process and thawing. Cell viability was assessed, in both fresh and thawed samples, by Hoechst 33258 staining. Computer-assisted sperm analysis (CASA) was used to determine the percentage of motile cells and to measure motility parameters in sperm samples. A significant decrease of head perimeter (12.56%) and area (17.90%) was detected from spermatozoa in fresh to thawed samples, indicating that cells do not recover the original size after the cryopreservation process. CASA was used to measure the percentage of motile cells (51.9%) and spermatozoa motility parameters such as curvilinear, straight line and angular path velocities, as well as beating cross frequency. This technique was employed in the fresh sperm samples but proteins present at the freezing medium (L-alpha-phosphatidylcholine) made impossible to use this last technique in thawed samples. When sperm viability was assessed by Hoechst staining, a significant decrease of approximately 15% (73.10 vs 58.26%) of alive spermatozoa was registered from fresh to thawed samples. The percentage of motile cells measured by CASA in fresh samples (51.9%) was lower than the percentage of alive cells determined by Hoechst stainning, suggesting the existence of different batches of spermatozoa in different stages of development, even during the eight to tenth weeks of treatment, when the highest sperm quality was found.     

Objective assessment of sperm motion characteristics of Malpura ram lambs raised under intensive management system in semiarid tropical environment

A study was conducted to evaluate the semen production and sperm motion characteristics of ram lambs by computer-aided semen analysis technique. Eight Malpura rams were raised under intensive management system and were trained for semen collection at a weekly interval from the age of 6 months. Rams were scheduled for semen collection at a weekly interval up to 1 year of age to assess their potential for semen production and objective evaluation of semen quality. The average age of ram lambs at the time of first ejaculation was 219 days ranging from 186 to 245 days. The age of ram lambs significantly (p < 0.05) influenced sperm concentration, sperm velocities, and beat frequency of spermatozoa, which were higher in 9–12-month-old compared to 6–9-month-old ram lambs. However, the effect of age was not significant on semen volume, percent motility, percent rapid, medium or slow motile spermatozoa, percent linearity, percent straightness, amplitude of lateral head displacement, percent elongation, and area of sperm head. The body weight of ram lambs was significantly (p < 0.01) and positively correlated (r = 0.46) with age. The results indicate that Malpura ram lambs of 9–12 months of age raised under the intensive management system in a semiarid tropical environment can produce good quality of semen.     

Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation

The aim of the study was to compare different types of movement pattern and velocities of stallion spermatozoa depending on cryopreservation during breeding and non-breeding season. Ejaculates were collected from four stallions during May (n = 24) and December (n = 24). Parameters of sperm movement were evaluated by computer-aided sperm analysis (CASA) system, and included percentages of motile spermatozoa, different patterns of motility, the velocity, linearity (LIN), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF). In winter the average percentages of motility were slightly higher compared to the breeding season in May (70.8 +/- 12.7% vs. 66.8 +/- 12.2%, respectively). Cryopreservation and thawing led to a significant decrease in the number of motile sperm to 11.3 +/- 5.8% in May and 15.6 +/- 7.0% in December. The pattern of motility was also changed. Detailed analysis by CASA demonstrated that cryopreservation resulted in a shift from the proportions of linear to more non-linear motile spermatozoa and to a significant increase of local motile and hyperactivated spermatozoa. Mean velocity of fresh motile spermatozoa differed between May and December (119.1 +/- 43.9 vs. 164.4 +/- 66.4 microm/sec, respectively; P < 0.05). Cryopreservation and thawing led to a slight increase of curvilinear velocity (VCL) and straight line velocity (VSL). The motility analysis has shown that the parameters BCF and ALH were highly correlated in stallion spermatozoa (r = -0.67; P < 0.001). The BCF of stallion spermatozoa was slightly reduced in the non-breeding season. Altogether, the influence of factors on the motility of stallion spermatozoa has the following rank order: cryopreservation (P < 0.0001) > stallion (P < 0.001) > season (P < 0.05).     

Evaluation of spermiation indices with multiple sperm collections in endangered sterlet (Acipenser ruthenus)

This study investigated the effects of multiple collections of sperm on endangered sterlet (Acipenser ruthenus) sperm functional parameters [spermatozoa motility and curvilinear velocity (VCL)] as well as on protein concentration and osmolality of seminal plasma. The average sperm volume and mean spermatozoa concentration per male were significantly altered with multiple collections. On the other hand, no significant effect of multiple collections on protein concentration of seminal plasma was observed. In all experimental groups, moderate impact of sequential collection on osmolality (p < 0.05) of seminal plasma was observed. Ninety to 100% of motile spermatozoa were observed at 15 s after activation, with an average VCL of 181.12 ± 19.10 μm/s. After 90 s, average VCL decreased to 130 ± 26 μm/s. Motility was maintained for up to 4 min. The maximum percentage of motile spermatozoa was observed after the third collection of sperm. The spermatozoa VCL increased significantly with subsequent collections. The results of this study provide new data on the effects of multiple collections on quantitative and qualitative parameters of sperm in sterlet. The data confirmed that the sequential stripping has no negative effect on the percentage of motility and spermatozoa velocity. This should be beneficial for the development of sterlet aquaculture programs.     

Prognostic Value of Various Spermatological Attributes as Predictors of Zona Binding and Zona Penetration of Buffalo (Bubalus bubalis) Semen

Twenty‐four ejaculates from six (four ejaculates each) Surti buffalo bulls aged 4–8 years were used to assess various attributes of spermatozoa influencing the zona‐binding and zona‐penetration tests. Ejaculates from each bulls were subjected to in vitro sperm‐‐zona binding and sperm‐‐zona penetration tests (four replicates per bull) using immature buffalo oocytes. The average number of spermatozoa bound per oocyte was 27.79 ± 5.90. The average number of spermatozoa penetrated per oocyte was 3.35 ± 0.64. The average number of zona‐bound and ‐penetrated spermatozoa differed significantly between animals. Significant difference (p < 0.05) was observed between the plasmalemma integrity as assessed by eosin‐‐nigrosin stain and hypo‐osmotic swelling (HOS) test. Furthermore, the percentage of cells positive for the HOS test, i.e. functional membrane integrity (51.25 ± 2.32) was significantly (p < 0.05) higher than hypo‐osmotic swelling‐Giemsa (HOS‐G) test, i.e. the subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (42.87 ± 4.56). The HOS test had significant correlations with plasmalemma integrity as measured by the vital stain, eosin‐‐nigrosin (r = 0.85, p < 0.05). The HOS‐G test also had significant correlation with plasmalemma integrity measured by vital stains such as eosin‐‐nigrosin (r = 0.90, p < 0.05) and fluorogenic stains [carboxyfluorescein diacetate (CFDA) and propidium iodide (PI); r = 0.92, p < 0.01] and HOS test (r = 0.93), acrosomal integrity (r = 0.86, p < 0.05) and mitochondrial membrane potential (r = 0.99, p < 0.01). The plasmalemma integrity (fluorogenic stain), functional membrane integrity (HOS test), subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (HOS‐G test) and mitochondrial membrane potential had significant (p < 0.05) correlation with sperm zona binding and penetration. The present study indicates that these parameters could represent important determinants of sperm quality influencing zona binding and penetration.     

Effects of Egg Yolk and Cooling Rate on the Survival of Refrigerated Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.     

Effects on boar semen quality after infection with porcine reproductive and respiratory syndrome virus: a case report

The effect of porcine reproductive and respiratory syndrome virus (PRRSV) on semen quality was examined in a group of 11 spontaneously infected boars in a commercial boar stud. Semen samples were collected 4 weeks prior to 4 weeks post-infection (wpi). Infection with PRRSV of the European genotype subtype 1 (EU-1) was verified by specific quantitative real-time polymerase chain reaction (RT-PCR) in 36% of the serum samples. All boars seroconverted before 4 wpi and remained in normal condition throughout the study. Comparison of the percentage of morphologically intact spermatozoa revealed an increase of acrosome-defective spermatozoa (P = 0.012) between −4 and 4 wpi. Significant deleterious effects on semen quality were detected for membrane integrity when semen had been stored for 2 days after sampling. Analysis of sperm subpopulations in a thermoresistance test on day 7 after sampling revealed alterations in the percentage of circular, progressively motile spermatozoa (P = 0.013), in the percentage of non-linear, progressively motile spermatozoa (P = 0.01), and on the amplitude of lateral sperm head displacement (P = 0.047). There was no difference in the incidence of mitochondrially active spermatozoa (P = 0.075). Investigation of routine production data between pre- and post-infection status showed no differences on ejaculate volume (P = 0.417), sperm concentration (P = 0.788), and percentage of motile spermatozoa (P = 0.321). This case report provides insights into a potential control strategy for PRRSV outbreaks in boar studs.     

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.     

Changes in Semen Quality in Estonian Holstein AI Bulls at 3, 5 and 7 years of Age

The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.     

Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa

Two experiments were conducted to test whether stallionand/or semen processing techniques influenced spermatozoal motility and acrosomal status following cold storage. Ejaculates from each of 18 stallions (N=54) were collected and split. In Experiment I, a skim milk-glucose extender (SKMG) was added to the semen following a 5, 15 or 30 minute delay post-collection. Following each delay, sperm were packaged at a final concentration of 25 million progressively motile sperm per ml (PMS/ml) in a commercially available skim milk-glucose extender (SKMG). In Experiment II, sperm were packaged at concentrations of 25, 50, and 75 million PMS/ml both in the presence and absence of seminal plasma (SP) utilizing SKMG and SKMG plus PBS, respectively. In both experiments, aliquots were cooled, stored, and the percentage of progressively motile and acrosome intact spermatozoa were determined at 24 and 48 hours post-collection. In Experiment 1, delayed dilution resulted in a lower recovery of PMS. In Experiment II, removal of SP resulted in higher percentages of PMS following cold storage. Increasing the concentration of spermatozoa during packaging decreased the percentage of PMS; however, removal of SP reduced the harmful effects on spermatozoa motility. These data suggest that reducing the time that spermatozoa remain in an undiluted state and removal of SP maximize recovery of progressively motile, acrosome-intact spermatozoa. In addition, individualizing the processing techniques for each stallion may enhance spermatozoal survival following cold storage.     

Evaluation of Spermatological Parameters Used to Predict the Fertility of Frozen Bull Semen

Kjxstad, H., E. Ropstad and K. Andersen Berg: Evaluation of spermatological parameters used to predict the fertility of frozen bull semen. Acta vet. scand. 1993,34,299-303.– Post-thaw motility, velocity and acrosome integrity of frozen semen were determined in 18 bulls with varying fertility (average non-return rates: 71.3 (± 2.8) - range: 65.2-75.7). Five semen straws were investigated from each bull. The average values for sperm motility (percentage motile spermatozoa), sperm velocity (graded from 0-3) and acrosome integrity (proportion of spermatozoa with intact acrosome) were 67.5%, 2.5 and 79.3%, respectively. Significant correlations were found between sperm motility and velocity, but not between sperm motility and acrosome integrity. Both sperm motility and velocity were significantly related to bull fertility. It was concluded that of the post-thaw semen characteristics investigated in this study these 2 parameters provided a reliable basis for prediction of bull fertility.