Correlations between heterospermic fertility and assays of porcine semen quality before and after cryopreservation

The relative fertilizing potential of frozen-thawed semen from four black and four white boars was determined following heterospermic insemination. A heterospermic index (HI) was computed for each of the 16 possible pairs of black and white boars. Correlation coefficients were computed between the HI and several in vitro tests of semen quality before and afttr cryopreservation of the semen. For the in vitro tests before cryopreservation, the HI were negatively correlated (-0.57) with spermatozoal motility before cooling the semen, but they were not correlated with spermatozoal motility after cooling to 5 degrees C. After freezing and thawing, the HI were correlated with the following in vitro tests: spermatozoal motility (0.50), spermatozoa with either normal or damaged apical ridges (0.31), spermatozoa with missing apical ridges (-0.51), spermatozoa filtered through sephadex columns (0.32), spermatozoa with acrosin-activity (0.38), percentage of maximal releasable glutamic oxalacetic transaminase (GOT) present extracellularly (0.54), spermatozoal intracellular GOT (-0.57), spermatozoa bound per zona-free hamster oocyte (0.64), and percentage of zona-free hamster oocytes penetrated (0.75). The HI were not correlated with the following in vitro tests after freezing and thawing: spermatozoa with normal apical ridges, damaged apical ridges and loose acrosomal caps, extracellular and maximal releasable GOT, and the number of penetrations per zona-free hamster oocyte. The multiple regression correlation coefficient between the HI and four selected variables from three in vitro tests was 0.94. This high correlation indicated that the fertilizing potential of the semen could be accurately predicted with four variables that appeared to measure different properties of the spermatozoa.     

Possible Effects of Metallosis on Spermatozoal Apoptotic Genes Expression in Individuals with Intramedullary Nailing Prosthesis

Seminal quality could be affected by metallosis caused by intramedullary nailing (IMN). Our objectives were to estimate metal ion levels in the seminal plasma of subjects with IMN, to determine their effects on semen parameters and on spermatozoal apoptotic gene expression, and to determine whether these expressed genes could be used as candidate biomarkers of seminal deterioration in individuals with IMN or not. Semen samples were collected from 60 subjects with IMN and 30 age-matched healthy controls. Seminal plasma contents of cobalt (Co), chromium (Cr), and molybdenum (Mo) were assayed. Spermatozoal Bcl-2 and Bax gene expressions were determined. Studied semen parameters were significantly lower in subjects with IMN for ≥5 years in relation to controls while the concentrations of Co, Cr, and Mo in the seminal plasma samples were significantly higher. There were significantly lower spermatozoal Bcl-2 expression, higher Bax expression, and lower Bcl-2/Bax ratio in subjects with IMN for ≥5 years than in controls. In subjects with IMN for ≥5 years, receiver operating characteristic (ROC) curve analysis of studied gene expressions and Bcl-2/Bax ratio were done showing priority of the ratio with 86.7 % sensitivity, 100 % specificity, 100 % positive predictive value, and 93.8 % negative predictive value at cutoff values ≤0.777. Co, Cr, and Mo metals are found at high concentrations in the seminal plasma of individuals with IMN leading to increased spermatozoal apoptotic activity. Spermatozoal Bcl-2/Bax ratio could be used as a candidate biomarker of reproductive disorders in individuals with intramedullary nailing.     

Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage

The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by < or = 30% of progressive motility prior to storage. Group 2 stallions ("poor coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by > or = 40% of progressive motility prior to storage. The sperm-rich portion of each ejaculate was divided into 4 aliquots. Two aliquots underwent standard processing for cooled transported semen and were examined after 24 and 48 h of cooling and storage in an Equitainer. The remaining two aliquots were diluted 1:1 with semen extender, then centrifuged at 400 x g for 12 min at room temperature. After centrifugation, approximately 90% of the seminal plasma was removed, and the sperm pellet was resuspended in extender to a final concentration of 25 to 50 x 10(6) sperm/mL. These aliquots were then packaged as for the non-centrifuged aliquots and examined after 24 and 48 h of storage. The spermatozoal motion characteristics in fresh semen and after 24 and 48 h of cooling and storage was determined via computer-assisted semen analysis. Centrifugation and partial removal of seminal plasma increased the percentage of progressively motile spermatozoa and limited the reduction in progressive spermatozoal motility of "poor cooling" stallions after 48 h of cooling and storage. Results of this study indicate that centrifugation and partial removal of seminal plasma is beneficial for stallions whose ejaculates have poor tolerance to cooling and storage with routine semen dilution and packaging techniques, especially if the semen is stored for > 24 h.     

Relationships among spermatozoal abnormalities and the selenium concentration of blood plasma,semen, and reproductive tissues in young bulls

The concentration of selenium (Se) was measured by instrumental neutron activation analysis in samples of blood plasma, semen, and reproductive and non-reproductive tissues obtained from each of 12 young bulls (8 Angus and 4 Simmental). Semen was collected by electro-ejaculation and used for measurements of the frequency of primary and secondary spermatozoal abnormalities. The mean Se concentration (μg/ml) of semen (± SD) was 0.461 (±0.223) compared to 0.061 (±0.014) for blood plasma. Tissue from the testis, caput epididymis and cauda epididymis had mean Se concentrations of from 2.5 to 2.7 μg/g compared to less than 1.8 μg/g in all other tissues except the pituitary gland (3.4 μg/g) and kidney cortex (6.9 μg/g).Correlations of the proportional incidence of spermatozoal abnormalities with Se concentrations in reproductive tissues, semen or blood plasma were low. Although Se may be concentrated in the testis and epididymis, the Se concentration was not related to spermatozoal abnormalities.     

The effect of anoxia on fowl spermatozoa: Recovery of fertilizing ability,motility, and cellular concentrations of potassium and ATP

Fowl semen when diluted in a glutamate-based medium without glucose, gradually lost its fertilizing ability during 4 hr of anaerobic incubation at 30°C. This incubation regime offered a system by which various in-vitro tests of spermatozoal viability could be assessed for their usefulness as monitors of fertilizing ability. Widely used tests such as spermatozoal enzyme leakage, dye exclusion, and morphology as assessed by light microscopy showed no change in spermatozoal status as the fertilizing ability declined. However the ability of sperm, during a short aerobic incubation to restore their motility and ATP and K⁺ concentrations, declined as did their fertilizing ability. When glucose was added to this re-aeration medium, spermatozoal motility, K⁺ and ATP concentrations, and fertilizing ability were restored to optimal levels. Thus the fertilizing ability of fowl sperm, following anaerobic storage at 30°C, appeared to be related to their ability to restore ATP and K⁺ concentrations and motility. An initial event in the loss of fertilizing ability was a loss in the ability of sperm to oxidise endogenous substrates. This could be restored by the addition of glucose.     

Seminal selenium concentrations and spermatozoal abnormalities in beef bulls

The relationship between seminal selenium (Se) concentration and spermatozoal abnormalities in 24 Angus and 12 Simmental bulls maintained on a Se adequate diet was studied. Two semen samples were collected by electroejaculation 50 days apart from each bull. Measurements of primary and secondary spermatozoal abnormalities, seminal Se concentration, and blood plasma Se concentration were determined at each semen collection. The mean (chi +/- SD ) Se concentration of semen (0.535 +/- 0.267) was approximately 8 fold greater than the Se concentration of blood plasma (0.069 +/- 0.066) and the values were similar for both collections. Spermatozoa concentration was correlated (r = 0.50; P<.01) with seminal Se concentration; however, seminal Se concentration was not highly correlated (P<.01) with primary spermatozoal abnormalities (r = -0.29) and secondary spermatozoal abnormalities (r = 0.16). This study indicates that the Se concentration of semen is high relative to blood plasma in bulls maintained on a Se adequate diet; however, the seminal Se concentration is not highly correlated with spermatozoal abnormalities.     

Inhibition of motility of bovine, canine and equine spermatozoa by artificial vagina lubricants

The effects of four vaginal lubricants on progressive spermatozoal motility were evaluated. Neat semen was exposed to 0, 5, or 10% (w/v) of H-R, sterile K-Y, nonsterile K-Y or Maxilube lubricating jellies for 10 min at 37 degrees C and then extended to 10x10(6) spermatozoa/ml. Spermatozoal motility was evaluated after 0, 1, 2, 4 and 6 or 8 h of incubation at 37 degrees C. For bovine spermatozoa, sterile K-Y jelly at 10% suppressed motility (P<0.05), but nonsterile K-Y, H-R and Maxilube jellies had no effect. Maxilube was toxic (P<0.01) to canine spermatozoa and is not recommended for use during collection or insemination of canine semen. Exposure of equine semen to 10% H-R jelly had no effect on spermatozoal motility, whereas 10% sterile K-Y, nonsterile K-Y or Maxilube jellies suppressed (P<0.05) motility. For all three species, the new, nonsterile K-Y jelly was no more deleterious to spermatozoal motility than the old, sterile K-Y jelly, and H-R jelly also was satisfactory. Fertility tests are required to determine the effect of these products on fertility.     

Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa

This study was conducted to compare the cooling rates and storage temperatures within equine semen transport containers exposed to different ambient temperatures, and to evaluate the ability of these containers to preserve spermatozoal motility following 24 h of storage under these conditions. In Experiment 1, nonfat dried milk solids, glucose, sucrose, equine semen extender was divided into seven 40-mL aliquots and loaded into seven different semen transport containers: Equitainer I, Equitainer II, Equitainer III, ExpectaFoal, Bio-Flite, Lane STS, and Equine Express. After containers were loaded, they were subjected to one of three ambient storage temperatures: 1) 22 degrees C for 72 h, 2) -20 degrees C for 6 h followed by 22 degrees C for 66 h, or 3) 37 degrees C for 72 h. Cooling rates and storage temperatures of semen extender in each container were monitored with thermocouples and a chart recorder. In Experiment 2, semen from each of three stallions (3 ejaculates per stallion) was diluted to 25 x 10(6) spermatozoa/mL with semen extender, divided into 40 mL aliquots and loaded into transport containers as in Experiment I. Containers were subjected to one of three ambient storage conditions: 1) 22 degrees C for 24 h, 2) -20 degrees C for 6 h, followed by 22 degrees C for 18 h, or 3) 37 degrees C for 24 h. After 24 h of storage, spermatozoal motion characteristics (percentage of motile spermatozoa; MOT, percentage of progressively motile spermatozoa; PMOT, and mean curvilinear velocity; VCL) were evaluated using a computerized spermatozoal motion analyzer. Significant interactions were detected among storage conditions and semen transport containers for the majority of the temperature endpoints measured. When exposed to temporary ambient freezing conditions, the lowest temperatures attained by samples in containers ranged from -2.8 to 0.8 degrees C. Lowest temperature samples attained was not correlated (P > 0.05) with spermatozoal motility under any ambient condition. However, time below 4 degrees C was highly correlated (P < 0.05) with a reduction in spermatozoal motility. Mean cooling rates from 20 degrees C to 8 degrees C did not correlate with spermatozoal motility, except when containers were exposed to temporary freezing conditions. No container cooled samples below 6 degrees C in 22 degrees C or 37 degrees C environments except for the ExpectaFoal, in which samples fell below 4 degrees C under all ambient conditions. Ambient temperature affected MOT, PMOT and VCL of semen stored in all containers (P < 0.05) except for the Equitainer II in which motion characteristics remained high and were similar among all ambient temperatures (P > 0.05). Results suggest that stallion semen may be able to tolerate a wider range of cooling rates and storage temperatures than previously considered safe.     

Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

Semen quality in dogs and the influence of a short-interval second ejaculation

Semen quality was examined in each of 65 known fertile dogs. Values were found to be similar to those previously published, although an apparent breed influence was demonstrated, with German shepherd dogs producing ejaculates of larger volume and greater total spermatozoal output than other breeds. A second ejaculate was collected from each dog with a mean interval of 63 min. The second samples had significantly lower values for the volume of the second fraction, the spermatozoal concentration and the total spermatozoal output. There were no differences for the percentage motility or the percentage of morphologically normal live spermatozoa. While there was no increase in semen quality of the second ejaculate, the technique may be useful since it results in the collection of approximately 70% more spermatozoa than a single ejaculate. These spermatozoa also had normal motility and morphology and could, therefore, be used for insemination or cryopreservation.     

Correlation between the spermatozoal characteristics and sperm penetration distance in polyacrylamide gel and bovine cervical mucus

Correlation between the spermatozoal characteristics and the sperm penetration distance in polyacrylamide gel was assessed, utilizing frozen thawed semen samples obtained from 6 bulls, and it was compared with the correlation between sperm penetration in bovine cervical mucus and spermatozoal characteristics. In vitro sperm penetration tests were performed with mucus and gel. The sperm penetration in gel and mucus was significantly and positively correlated with post-thaw motility (r=0.81; r=0.89:P<0.01) and acrosome integrity (r=0.88; r=0.94:P<0.01). A significant negative correlation with abnormal spermatozoa (r=-0.84;r=0.83:P<0.01) was observed. Both sperm concentration and post-thaw live spermatozoa were not significantly correlated. A significant multiple regression between sperm penetration and the spermatozoal characteristics both in gel (R2=0.87; F=40.27; P<0.01) and mucus (R2=0.91; F=60.48; P<0.01) was observed. The major spermatozoal characteristics determining the capacity of spermatozoa to penetrate gel were post-thaw motility, percentage of abnormal spermatozoa and acrosome integrity. The acrosome integrity has a more significant contribution. The correlation established with sperm penetration in gel was very similar to that of sperm penetration in mucus. The utility of gel as a mucus substitute in in vitro sperm penetration tests was discussed.     

Effect of electro-ejaculation frequency on semen characteristics in the ferret (Mustela putorius) (a)

Ten sexually mature male ferrets were electro-ejaculated at 1, 3 and 5-day intervals to ascertain the effects of ejaculation frequency on semen quality. Semen volume, spermatozoa concentration, spermatozoal motility, as well as the number of stimuli required to obtain an ejaculation were recorded. No significant differences in these parameters were noted between the test intervals for 1 to 3-year-old males. The data indicated that male ferrets from 1 to 4 years of age may be ejaculated as frequently as once per day for short periods of time without any apparent adverse effects on semen quality.     

Influence of bacteria and gentamicin on cooled-stored stallion spermatozoa

This study investigated effects of bacteria from the genital tract of horses and the effect of gentamicin in semen extender on spermatozoal function in cooled-stored stallion semen. Semen was collected from healthy stallions and processed with a milk-based extender with or without gentamicin (1g/l). Pseudomonas (Ps.) aeruginosa, Staphylococcus (St.) aureus, Streptococcus (Sc.) equi subsp. equi (Sc. equi), Sc. equi subsp. zooepidemicus (Sc. zooepidemicus), Sc. dysgalactiae subsp. equisimilis (Sc. equisimilis) or culture medium alone (control) were added. Immediately after addition of bacteria and after storage at 5 degrees C for 24, 48 and 72h, motility, velocity and membrane integrity of diluted semen were determined with a CASA system. After 24h, semen with Ps. aeruginosa and Sc. equisimilis showed significantly lower motility and velocity compared to all other groups; after 72h these differences still existed for Ps. aeruginosa (p<0.05). The percentage of membrane-intact spermatozoa was significantly lower after 24h of storage in spermatozoa incubated with Sc. equisimilis and after 72h with Sc. equisimilis and Ps. aeruginosa. Addition of gentamicin to extender resulted in decreased motility and velocity in semen without addition of bacteria and did not improve motility parameters in semen with bacteria added. In conclusion, certain bacteria may have detrimental effects on semen quality during cooled-storage. These effects are not reduced by addition of gentamicin. Gentamicin can negatively affect spermatozoal function in extended semen during cooled-storage and therefore, optimal concentrations have to be tested for the respective extender medium.     

Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires

This study determined the relationship between two measures of field fertility of 11 high-use Australian artificial insemination (AI) dairy bulls and thirty standard laboratory assessments of spermatozoal post-thaw viability.The two measures of field fertility used, conception rates (cCR) and non-return rates (cNRR), were both corrected for all major non-bull variables. Sperm viability assessments were conducted on semen collected within the same season as that used to derive the field fertility estimates. These assessments measured sperm concentration, motility, morphology and membrane integrity at thawing, after 2h incubation and after the swim-up sperm selection procedure. Derivations of these measures and in vitro embryo fertilizing and developmental capacity were also determined. The Genstat Statistical Package [Genstat 5 Release 4.2 Reference Manual, VSN International, Oxford, 2000] was used to conduct an analysis of variance on the viability parameters across semen straws and bulls, and to calculate the strength of correlation between each semen parameter, cNRR and cCR in a correlation matrix. Step forward multiple regression identified the combination of semen parameters that were most highly correlated with cCR and with cNRR.The sperm parameters identified as being most predictive of cCR were the percentage of morphologically normal sperm immediately post-thaw (zeroNorm), the number of morphologically normal sperm after the swim-up procedure (nSuNorm), and the rate of zygote cleavage in vitro (Clv); the predictive equation formed by these parameters accounted for 70% of variance. The predictive equation produced for cNRR contained the variables zeroNorm, the proportion of membrane intact sperm after 2h incubation at 37 degrees C (twoMem) and Clv and accounted for 76.5% of the variation. ZeroNorm was found to be consistent across straws and semen batches within-bull and the sperm parameter with the strongest individual predictive capacity for both cCR (P=0.1) and cNRR (P=0.001). Post-thaw sperm parameters can be used to predict field fertility of Australian dairy sires; the calculated predictive equations are particularly useful for identifying and monitoring bulls of very high and very low potential fertility within a group.     

Evaluation of fresh and frozen-thawed fowl semen by flow cytometry

The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.     

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.     

Enzyme leakage during cryopreservation of ram spermatozoa

Spermatozoal motility and enzyme leakage during cryopreservation of ram spermatozoa was evaluated in the presence and absence of antifreeze proteins (AFP). Loss of spermatozoal motility due to the cryopreservation process was reduced by 10% in the presence of AFP. Samples of the diluted semen at various stages of the cryopreservation process were assayed for aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), acid phosphatase (ACP) and lactate dehydrogenase (LDH). The levels of AST, ALT and ACP were low in ram spermatozoa and were not considered suitable for the objective measurement of spermatozoal damage. ALP leaked during the cooling and freezing process, whereas LDH leakage was prevalent during cooling, freezing and post-thaw incubation of spermatozoa. Changes in ALP and LDH could be used as marker enzymes in the development of semen processing protocols and of semen diluents.     

Radiographic contrast medium for uterine insemination in the bitch, and its effect upon the quality and fertility of fresh dog semen

Four ejaculates were collected from each of 6 adult male beagle dogs. The second fraction was divided into 3 aliquants which were then diluted with physiological saline, an isoosmolar solution of sodium/meglumine diatroate, and an iso-osmolar solution of iohexol. The diluted samples were incubated at 39 degrees C and evaluated at 0, 60, 90, 120, 240 and 360 min after dilution. A variety of assessments was made, including, spermatozoal motility, spermatozoal morphology, and acrosorne status. The practicality of using 1.0 ml of iso-osmolar contrast medium combined with radiographic examination was evaluated as a method of confirming accurate placement of a transcervical uterine catheter by injecting contrast after positioning the catheter in 4 beagle bitches. The effect of the procedure on fertility was assessed using 5 greyhound bitches which were inseminated with fresh semen and in which pregnancy was monitored using diagnostic B-mode ultrasound imaging. There was no significant difference between physiological saline and the sodium/meglumine diatroate solution upon semen quality, while the iohexol solution produced a significant reduction in spermatozoal motility and morphology. No adverse clinical effects were observed when contrast medium was administered into the uterus to either group of bitches. A subjective assessment of radiographic quality showed that the sodium/meglumine diatroate solution, which contained twice the iodine concentration of the iohexol solution, produced significantly greater radiopacity and was radiographically more useful than the iohexol solution. The sodium/meglurnine diatroate solution had no adverse effect upon the fertility of dog semen, and all bitches that were inseminated with this technique conceived and maintained the pregnancy to term. Litter size was considered to be normal for the breed. Small volumes of an iso-osmolar solution of sodium/meglumine diatroate may be useful for ensuring correct placement of transcervical catheters prior to artificial insemination in the bitch.     

Proteolytic enzymes stimulate human spermatozoal motility and in vitro hamster egg penetration

Addition of chymotrypsin or trypsin decreased the viscosity of highly viscous semen of subfertile patients. Besides that spermatozoal motility (in 50-80% of the case) and in vitro fertilizing ability was measured with zona-free hamster ova (in 35% of the cases) were increased of both normal and highly viscous semen.