Higher Percentage Of Motile Sperm Research Articles

Effects of pharmacological doses of niacin on subacute glucocorticoid-induced testicular damage in rats.

Glucocorticoid excess adversely affects male reproduction. This study evaluates effects of pharmacological doses of niacin on testicular structure and function in dexamethasone-treated rats. Adult rats (48) were randomly assigned to 6 equal groups: (1) Negative control (NC): normal rats; (2) Positive control (PC): dexamethasone at 7 mg/kg/day by intraperitoneal injections for 7 days; groups 3-6 (N50, N100, N200, and N400): dexamethasone and concomitant treatment with niacin at 50, 100, 200, and 400 mg/kg/day by oral gavages. Testicular weight and volume of PC rats were significantly lower than the NC group (p < .05). Testicular volume of rats in the N50 and N200 groups was statistically similar to the NC group. Significant decreases in serum testosterone with a slight LH increase were detected in the PC group. Nacin at 50 mg/kg reversed serum testosterone to NC levels and increased serum LH concentration. Niacin only slightly increased epididymal spermatozoa number while all groups of niacin-treated rats had significantly higher percentages of motile spermatozoa compared with the PC group. Hypospermatogenesis, germ cell degeneration and depletion, epithelial vacuolization, and degenerated Leydig cells were observed in PC rats. Lesions were relatively milder in niacin-treated rats. Johnsen scores were also significantly higher in niacin-treated rats. Niacin reduced apoptosis as shown by TUNEL assay. In conclusion, niacin administration at pharmacological doses dose-dependently ameliorates the destructive effects of dexamethasone on sperm motility, Johnsen score, and testicular cell apoptosis in rats with the latter can be considered a decisive mechanism for its positive effects on testis.

The endocrine disruptor 17β-trenbolone alters the relationship between pre- and post-copulatory sexual traits in male mosquitofish (Gambusia holbrooki)

It is now well-established that reproduction in wildlife can be disrupted by anthropogenic environmental changes, such as chemical pollution. However, very little is known about how these pollutants might affect the interplay between pre- and post-copulatory mechanisms of sexual selection. Here, we investigated the impacts of 21-day exposure of male eastern mosquitofish (Gambusia holbrooki) to a field-realistic level (average measured concentration: 11 ng/L) of the endocrine-disrupting chemical 17β-trenbolone (17β-TB) on pre- and post-copulatory reproductive traits. We examined male reproductive behaviour by testing the time spent near a female behind a partition, as well as the number of copulation attempts made, and the time spent chasing a female in a free-swimming context. Sperm traits were also assayed for all males. We found that exposure of male fish to 17β-TB altered the relationship between key pre- and post-copulatory reproductive traits. Furthermore, 17β-TB-exposed males had, on average, a higher percentage of motile sperm, and performed fewer copulation attempts than unexposed males. However, there was no overall effect of 17β-TB exposure on either the time males spent associating with or chasing females. Taken together, our findings demonstrate the potential for chemical pollutants to affect both pre- and post-copulatory sexual traits, and the interplay between these mechanisms of sexual selection in contaminated wildlife.

Effects of salinity, temperature and pH on the motility in Siganus guttatus spermatozoa

The effects of salinity, temperature and pH on the activity in the spermatozoa of Siganus guttatus were studied. Milt were collected from 8 wild mature males that came from a commercial hatchery. Sperm motility was measured with a VHS video-recorder and a video camera connected to a microscope. The results showed that most of the sperm were highly motile in 400-700 mM NaCl, the longest duration of sperm motility recorded in 600mM NaCl and the duration was 788.0±16.9 s. Sperm was not motile when suspended at pH 4, sperm motility was observed at pH 5, there was a relatively higher percentage of motile sperm in solutions at pH 5-8 (above 80%), the duration was the longest and achieved (769.0±19.8) s when pH was 8. The motility and duration increased within 10-25ºC and decreased at the range of 25-40ºC, the highest motility and the longest duration were all appeared at 25 ºC, they were (91.0±1.4) % and (725.0±21.2) severally. Optimum salinity, temperature and pH for S.guttatus reproduction was 600mM NaCl, 25 ºC and 8 respectively.

34 The effect of dilution method of beagle dog semen on the survival rate of cryopreserved spermatozoa after thawing

The successful cryopreservation of spermatozoa of the beagle dog for AI is essential for the establishment of the genetic banks of drug detection dogs. The beagle dog is widely used for drug testing and chosen for breeding by breeders. However, the use of cryopreserved beagle semen is limited by the lower number of offspring of dog species. In this study, 3 highly trained beagle dogs were chosen and their semen was cryopreserved for the next generation. The effects of dilution methods of beagle semen were tested using a direct dilution method at RT and a 2-step dilution method at 5°C. As a control group, the effects of a direct dilution method of semen on the percentage of motile sperm and progressive motility were analysed by computer-assisted semen analysis system (SAIS, Korea), and abnormality of spermatozoa was examined by Diff Quik staining. A total of 9 samples from 3 dogs were extended in 4% glycerol containing Tris-egg yolk diluents at approximately 22 to 25°C. The diluted semen was cooled to 5°C within 2h. The packed 0.5-mL straws were placed 5cm above the surface of LN for 10min and then plunged in. A 2-step dilution method was conducted using the same procedures of freezing, but the first dilution was done with glycerol-free diluent. After cooling to 5°C within 2h, the second diluent with 8% glycerol was added to the same volume of diluted semen at 5°C and stabilised for 1h. After thawing for 45s at 37°C, the semen from the 2-step dilution method showed the higher percentage of motile sperm (65.4±6% v. 45.3±8%; P&amp;lt;0.05) and progressive motility (41.6±5.3% v. 32.3±3.7%; P&amp;lt;0.05). However, the abnormalities between groups showed no differences. The results suggest that the optimal method for freezing beagle dog spermatozoa is a 2-step dilution process that consists of the first dilution at RT and the second dilution with glycerol at 5°C into diluted semen.

Changes of sperm morphology, volume, density, and motility parameters in northern pike during the spawning period.

Sexually mature males (BW = 1600 ± 150g and TL = 235 ± 30mm) of northern pike (Esox lucius L.) were randomly selected from a pond to record changes in their sperm quality parameters (spermatozoa morphology, sperm volume, density, and motility parameters) during the spawning season. The morphological and motility parameters changed significantly during the reproductive season with following trends. Only, head width was not changed during the spawning season. The longest spermatozoa and its flagellar length were found at the middle of spawning period (TL = 38.24 ± 0.37μm and 35.14 ± 0.26μm) and shortest at the beginning of spawning period (TL = 34.81 ± 0.29μm and 32.53 ± 0.18μm). Other morphological characters were always the lowest at the beginning of spawning period. Sperm volume was changed from 0.33 ± 0.3ml in February, 0.43 ± 0.2ml in March to 0.24 ± 0.1ml in April, and density from 16.2 ± 0.2 × 109 spermatozoa ml-1 in February, 19.4 ± 0.2 × 109 spermatozoa ml-1 in March to 4.8 ± 0.2 × 109 spermatozoa ml-1 in April. Same sperm velocity was observed in all spawning terms at 10 and 20s after activation. Higher velocity was found at 30 and 40s after activation in sperm collected at the middle and the end of spawning period. Significantly, higher percentage of motile sperm was observed at 20, 30, and 40s after activation in sperm sampled at the end of spawning period. This study supports the hypothesis that longer spermatozoa swim faster.

Improved sperm kinematics in semen samples collected after 2 h versus 4–7 days of ejaculation abstinence

Does a short abstinence period of only 2 h yield spermatozoa with better motility characteristics than samples collected after 4-7 days? Despite lower semen volume, sperm concentration, total sperm counts and total motile counts, higher percentages of motile spermatozoa with higher velocity and progressiveness were detected in samples obtained after 2 h. Most studies that have assessed the effect of abstinence periods on sperm motility parameters in men with a sperm concentration below 15 million/ml have detected a higher percentage of motile spermatozoa in samples obtained after short abstinence periods. Studies of men with sperm concentrations above 15 million/ml have reported significantly decreased motile sperm counts after 24 h of abstinence compared with longer abstinence periods. This study had a controlled repeated-measures design based on semen samples from 43 male partners, in couples attending for IVF treatment, who had a sperm concentration above 15 million/ml. Data were collected between June 2014 and December 2015 in the Fertility Unit of Aalborg University Hospital (Aalborg, Denmark). Participants provided a semen sample after 4-7 days of abstinence followed by another sample after only 2 h. For both ejaculates, sperm concentration, total sperm counts, motility groups and detailed kinematic parameters were assessed and compared by using the Sperm Class Analyzer (SCA) computer-aided sperm analysis system before and after density gradient selection. The laboratory's local manual method (Makler chamber) was used for comparison. The second raw ejaculate demonstrated lower semen volume (P < 0.0001), sperm concentration (P = 0.003) and sperm counts in all motility sub-groups (P < 0.001) but higher percentages of spermatozoa with higher velocity (P < 0.01), progressiveness (P < 0.001) and hyperactivation (P < 0.001), compared with the first raw ejaculate. The first ejaculate in this study was also used for the IVF/ICSI treatments and therefore only patients with a semen volume ≥2 ml and concentration ≥15 million/ml were included. Further validation in large prospective randomized controlled trials, more purposely directed at normozoospermic males with partners having problems conceiving when there appears to be no female factor, is needed to confirm the potential advantage of using a second semen sample in improving fertilization and pregnancy rates in assisted reproduction. Despite the significantly lower semen volume, sperm concentration and total sperm counts in all motility sub-groups, the significantly higher percentage of spermatozoa with better motility characteristics (velocity, progressiveness and hyperactivation) in the second ejaculate, may provide and allow for a simpler and more effective selection of higher quality spermatozoa. This could prove to be an advantage for ART procedures such as intracytoplasmic sperm injection where a large number of spermatozoa is not needed. It can also be speculated that pooling two consecutive ejaculates obtained after 4-7 days and after 2 h, could be an advantage for intrauterine insemination where a large number of motile spermatozoa are needed. This study was supported by internal grants from the Department of Health Science and Technology, Faculty of Medicine, Aalborg University (Aalborg, Denmark). The SCA® was provided by a grant from 'Ferring Pharmaceuticals' to Aalborg University Hospital (H.I.N). G.V.D.H. is an external senior scientific consultant to Microptic S/L (Barcelona, Spain). H.A. has provided scientific input and presentations for Microptic S/L (Barcelona, Spain) on several occasions. All other authors declare no conflict of interest. N/A.

Cryopreservation of bison epididymal sperm: A strategy for improving post-thaw quality when collecting sperm in field conditions

This study was conducted to optimize the cryopreservation of epididymal bison sperm harvested in the field. In the first experiment, epididymal bison sperm were treated with or without seminal plasma (n = 6) and cooled to 5 °C over 2 hours. In a separate experiment, glycerol was added at different times and sperm was held at 5 °C for different periods of time before cryopreservation (n = 11). In addition, epididymal sperm frozen with and without seminal plasma (n = 6) and after 4, 24, and 48 hours (n = 5) of equilibration at 5 °C, were evaluated for their in vitro fertilizing ability. Post-thaw motility of bison epididymal sperm was similar when cryopreserved with or without seminal plasma or when glycerol was added at either 0, 4, 24, or 48 hours before freezing (P > 0.05). However, sperm incubated at 5 °C for 24 hours before freezing exhibited higher percentages of motile sperm (44% vs. 35% for 4 hours or 48 hours, P < 0.05). Fertilization rates of bison oocytes were not different for any treatments. Chilling the whole epididymis for 24 or 48 hours resulted in complete loss of sperm viability. In conclusion, bison epididymal sperm can be chilled outside of the epididymis for at least 48 hours before cryopreservation without compromising post-thaw sperm motility providing flexibility for technicians performing field collections.

Seasonal variation of sperm quality in Pacific oyster (Crassostrea gigas)

Seasonal changes of sperm quality which can affect sperm biological parameters throughout the breeding period, have been little studied in mollusc species. Controlling gamete quality would aid the management of gametes in hatcheries and the development of selection programs. The aim of the present study was to describe the changes in sperm quality of wild Pacific oysters through the spawning season by comparing sperm parameters at the beginning (May), middle (July) and end (October) of this period using a panel of bio-descriptors. These parameters were studied over the 2014 breeding season based on shed sperm collected after serotonin injection of wild breeders. A significantly higher percentage of motile sperm was observed at the end of the spawning season (+78% relative to the value observed at the beginning) althought a lower total number of spermatozoa was collected (−59%). The mean condition index of oysters, however, was no different between the three sampling dates. For intratesticular sperm, the increase of the percentage of motile sperm and Velocity of the Average Path (VAP) in relation to time post activation was not significantly different among sperm sampling periods, suggesting that the kinetic of the sperm maturation process was similar. Furthermore, the mean VAP observed on shed sperm did not change through the spawning season. The sub-continuous gametogenesis of Pacific oyster can help to explain why only limited consequences of sperm ageing are observed in this species. Furthermore, the effects of sperm ageing may depend on the annual reproductive pattern of Pacific oyster. Statement of relevanceThis study showed the effect of sperm ageing on sperm quality parameters. This knowledge is useful for aquaculture and would support the recent trends of mollusc farming, allowing a better management of the gametes in hatchery.

Sperm mitochondrial membrane potential and motility pattern in the Holstein bull semen positive for heparin binding proteins

Present study was aimed to evaluate sperm motility and mitochondrial membrane potential (MMP) in relation to the presence of heparin binding proteins (HBPs) in semen. Semen was collected from 17 Holstein Friesian breeding bulls. Seminal plasma and sperm membrane proteins were isolated by precipitation method; HBPs were isolated using heparin-sepharose affinity chromatography. SDS-PAGE was performed and bulls were categorized into 3 groups based on presence/absence of 24 and 30 kDa HBPs. Computer-assisted semen analysis revealed that fresh ejaculates of bulls positive for 30 kDa HBP had 4.24 and 14.18% of higher total motile sperm than semen with 24 kDa and negative for HBPs. Semen with 30 and 24 kDa had 18.72 and 12.34% of higher number of sperm with progressive motility and velocity than semen devoid of HBPs. Simultaneous assessment of plasma membrane integrity (PMI) and MMP by fluorescence staining revealed that post-thaw sperm of semen with 30 kDa HBP had 6.28 and 12.79% of higher intact PMI. Further, sperm of semen with 30 kDa HBP had 4.61 and 10.50% of higher MMP, which had travelled an extra distance of 9.71 and 4.95 mm through cervical mucus than semen with 24 kDa and devoid of HBPs. The mucus penetration distance showed a strong positive correlation with high MMP, PMI, sperm motility and velocity. It is to be concluded that HBPs were able to sustain higher percentage of motile sperm with progressive motility and MMP which was depicted by more mucus penetration distance.

Optimizing sperm cryopreservation in great scallop (Pecten maximus)

A basic cryopreservation protocol for great scallop sperm was recently published, with which thawed sperm survival remained limited to 25% of control values (Suquet et al. 2014). We designed here a set of five experiments to test ways of improving thawed sperm survival and storage by studying the effects of (1) cryoprotectant concentration; (2) sugar incorporation in the extender; (3) egg yolk addition to the extender; (4) sperm dilution rate; and (5) storage capacity of thawed sperm at 4 degrees C. A sixth experiment was then performed to compare rearing performances of larvae produced using thawed sperm from the optimal protocol defined in the present study with those produced using fresh sperm. During the first experiment, a significantly higher percentage of motile spermatozoa was recorded after thawing when the treatment had included 15 or 20% polyethylene glycol (PEG). In the second experiment, no significant improvement in post-thaw sperm motility was recorded when trehalose or glucose was added to the extender. In the third experiment, adding 10 or 20% egg yolk to the extender significantly decreased the percentage of motile sperm. During the fourth experiment, significantly higher percentages of motile sperm were observed for dilution rates ranging from 3 : 1 to 1 : 1 than for higher dilution rates. In the fifth experiment, a significant decrease in the percentage of post-thawed sperm was observed for storage durations of 30 and 60 min, relative to shorter durations. During the last experiment, similar rearing performances were observed between larvae produced using fresh and thawed sperm, up to 9 days post fertilization. In conclusion, the high survival of sperm recorded after thawing (67% of the control level) and the high rearing performances of progenies obtained using thawed sperm validate the use of the present improved cryopreservation protocol for sperm cryobanking in great scallop.

Cholesterol-loaded cyclodextrin in fresh goat sperm improves cryosurvival rates

This study evaluated the effect of adding different concentration of cholesterol-loaded-cyclodextrin (CLC) on sperm quality after thawing. Thirty ejaculates were diluted, centrifuged and resuspended to 120 million cells/mL in a Tris diluent. Semen was treated with 0 (Control), 0.75, 1.5, 3.0, 4.5, 6.0 or 7.5 mg of CLC. Then, the samples were cooled at 4 °C for 2 h, diluted with Tris-egg yolk and 2% glycerol, packaged into 0.5 mL straws, frozen in liquid nitrogen (N2 ) vapor for 20 min before being plunged into N2 . Straws were thawed at 37 °C for 30 sec, and evaluated for thermal resistance test (TRT); progressive motility using CASA; hypoosmotic test and for binding capacity of sperm to perivitelline membrane (PM). The variables were determined using ANOVA at 5% probability. Higher percentage of motile sperm were maintained after thawing and TTR when 0.75 mg CLC was added, evaluated at 0, 60 and 120 min of incubation (50.4, 33.8 and 22.5%, respectively) compared to other treatments (P<0.05). The percentage of coiling was higher in sperm treated with 6.0 and 7.0 mg of CLC than other treatments (P<0.05). Addition of 0.75 mg CLCs also resulted in more sperm binding to the PM after cryopreservation than control sperm (166 vs 65; P<0.05). However, when the spermatozoa binding potential was determined on a motile sperm basis by dividing the average number of spermatozoa bound to PM for each bucks by the percentage of motile spermatozoa, CLC treatment provided higher binding efficiency (1.52) than control (1.00; P<0.05). In conclusion, CLCs improved the percentage of post-thaw of motility in caprine sperm as well as increased the number of sperm that bind to PM. Addition of 0.75 mg of CLC to caprine sperm prior to cryopreservation improved the quality sperm motility for up to 2 h.

Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation

This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the experiments, the treated sperm were incubated for 15min at 22°C to allow for the incorporation of cholesterol or cholestanol. In each experiment, treated sperm incubated for 15min at 22°C to allow for incorporation of cholesterol or cholestanol. The samples were then diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5°C over a 2h period. Loaded into 0.25ml polyvinylchloride straws, frozen in liquid nitrogen vapor for 10min, and then plunged into liquid nitrogen until further use. Higher percentages of motile sperm and viable cells were achieved after thawing for stallion sperm treated with CLC and CnLC compared to control (P<0.05). Addition of CnLC also resulted in more number sperm binding to chicken egg perivitelline membrane (CEPM) after cryopreservation than cholestanol and control sperm (P<0.05). In conclusion, CnLC and CLC improved the percentage of post-thaw motility of equine sperm and CnLC provided greater binding efficiency.

Sperm competition in humans: mate guarding behavior negatively correlates with ejaculate quality.

In species where females mate with multiple males, the sperm from these males must compete to fertilise available ova. Sexual selection from sperm competition is expected to favor opposing adaptations in males that function either in the avoidance of sperm competition (by guarding females from rival males) or in the engagement in sperm competition (by increased expenditure on the ejaculate). The extent to which males may adjust the relative use of these opposing tactics has been relatively neglected. Where males can successfully avoid sperm competition from rivals, one might expect a decrease in their expenditure on tactics for the engagement in sperm competition and vice versa. In this study, we examine the relationship between mate guarding and ejaculate quality using humans as an empirical model. We found that men who performed fewer mate guarding behaviors produced higher quality ejaculates, having a greater concentration of sperm, a higher percentage of motile sperm and sperm that swam faster and less erratically. These effects were found independent of lifestyle factors or factors related to male quality. Our findings suggest that male expenditure on mate guarding and on the ejaculate may represent alternative routes to paternity assurance in humans.

Effect of sampling location, release technique and time after activation on the movement characteristics of scallop (Pecten maximus) sperm

Sperm characteristics of scallops have not been well described in the scientific literature. The effects of sperm release technique (thermal shock versus serotonin injection), of sperm collection technique (testis sampling ver- sus serotonin injection), of sperm sampling location along the genital tract, of in vitro sperm maturation, and of time post activation on scallop sperm characteristics were assessed in the present work. Whatever sperm release technique used, no significant differences were observed regarding the percentage of motile spermatozoa and the velocity of the average path (VAP). Compared to testicular sperm, a higher percentage of motile spermatozoa, VAP and intracellular adenosine triphosphate (ATP) content were observed for sperm shed after serotonin injection. From the distal part of testes up to the gonopore, an increase of the percentage of motile spermatozoa and VAP was assessed, suggesting a sperm 'maturation process' along the genital ducts. A higher increase in the percentage of motile sperm was recorded during a 5 min incubation of testicular sperm in seawater containing 2 mM serotonin and seawater containing 10 mM caffein compared to seawater (control). In addition, a higher VAP was assessed, incubating testicular sperm in caf- fein, compared to control or serotonin. Then, the percentage of motile spermatozoa, VAP and intracellular ATP content exhibited a progressive reduction during the 10 h swimming period. Mean values of the percentage of motile spermato- zoa, VAP, sperm track linearity (LIN) and intracellular ATP content recorded at the beginning of the movement period for sperm samples collected after intragonadal serotonin injection, were 82 ± 7%, 162 ± 15 µ ms −1 ,0 .33 ± 0.12 and 212 ± 133 nmol ×10 −9 spermatozoa (n = 9 males), respectively. The present study confirms the existence of a sperm maturation process along scallop genital ducts. In addition, the cessation of scallop sperm movement can be explained by the exhaustion of ATP content at the end of the movement phase.

242 PROTECTIVE EFFECT OF VITAMIN E TREATMENT IN HEAT-STRESSED BOS TAURUS BULLS

Bos taurus bulls, when raised under tropical conditions, are highly susceptible to heat stress, which leads to impaired semen quality, leading to significant economical losses because, in these regions, the reproductive mounting season occurs mainly during the summer. Previous studies have indicated that oxidative stress (i.e. attack by reactive oxygen species) may be the main mechanism of sperm damage in such conditions. Therefore, treatment with antioxidants may be an important alternative to improve semen quality in heat-stressed B. taurus bulls. The objective of the present study was to evaluate whether the treatment with vitamin E, an important antioxidant, could improve sperm quality in insulated bulls. Towards this aim, eight adult Holstein bulls were submitted for semen collection, and the sperm was submitted for motility evaluation by computer-assisted sperm analysis (Ivos, Hamilton Thorne Inc., Beverly, MA, USA), examination of membrane and acrosomal integrity (eosin/nigrosin and fast green/bengal rose stain, respectively), mitochondrial activity (diaminobenzidine stain; full mitochondrial activity or no mitochondrial activity), and sperm susceptibility to oxidative stress (thiobarbituric acid-reactive substances). Bulls were then insulated (testicles covered in a thermal bag for 3 days) and randomly assigned to two treatment groups: no vitamin E (placebo) and vitamin E (subcutaneous injection of 3000 IU of α-tocopherol each of 10 days). Subsequent semen analysis was performed 1 and 60 days after the insulation. Statistical analysis was performed with SAS (SAS Institute Inc., Cary, NC, USA) repeated-measures ANOVA, and significance of P &lt; 0.05 was adopted. No differences were found on any of the variables before insulation. One day after insulation, animals treated with vitamin E showed a lower percentage of static sperm and a higher percentage of motile sperm when compared with animals treated with the placebo (28 and 63% v. 56 and 34%, respectively; P &lt; 0.05). Also at this time, sperm susceptibility to oxidative stress was lower in animals treated with vitamin E (vitamin E: 410 ng/106 sperm; no vitamin E: 1760 ng/106 sperm; P &lt; 0.05). Sixty days after insulation, sperm susceptibility to oxidative stress was still lower in animals treated with vitamin E when compared with the placebo group (1176 and 192 ng/106 sperm, respectively; P &lt; 0.05). However, no differences were found on the other variables. Results indicate that vitamin E, an antioxidant whose main function is protection of the plasma membrane, may be an alternative to avoid the acute deleterious effects of the heat stress in B. taurus bulls raised under tropical conditions. In addition, even with no heat stress involved, vitamin E treatment may provide constant protection, increasing the resistance of the sperm against the reactive oxygen species.

Characteristics of Siberian sturgeon and sterlet sperm motility parameters compared using CASA

Computer Assisted Sperm Analysis (CASA) was used to evaluate and compare the sperm motility parameters of Siberian sturgeon, Acipenser baerii Brandt (n=15), and sterlet, Acipenser ruthenus L. (n=15). Analysis indicated a higher percentage of motile sperm (MOT) in the sterlet semen (44.8%) than in that of the Siberian sturgeon (41.3%). The sperm of the two species studied were similar with regard to velocity, and similarities were also noted with regard to average path velocity VAP (97.6 and 102.4 µm s -1 ), curvilinear velocity VCL (121.9 and 119.9 µm s -1 ), and straight line velocity VSL (82.5 and 86.8 µm s -1 ). Using the CASA system permitted making precise evaluations of the sperm motility of the two fish species studied, and thus also permitted determining the potential suitability of the semen for use in sturgeon reproduction.

Semen cryopreservation for the creation of a Spanish poultry breeds cryobank: Optimization of freezing rate and equilibration time

A sperm cryopreservation protocol requiring dimethylacetamide (DMA, 6%) as a cryoprotectant was optimized via assays involving different prefreezing equilibration times (1, 10, 30, 60, and 120 min at 5°C) and different freezing rates achieved by the following: 1) using nitrogen vapor to reduce the temperature from 5°C to -85°C at 10°C/min (slow freezing rate); 2) using a biological freezer unit in a 2-step method to reduce the temperature from 5°C to -35°C at 7°C/min and then from -35°C to -140°C at 60°C/min (medium freezing rate); or 3) using a biological freezer unit in a 1-step freezing method to reduce the temperature from 5°C to -180°C at 60°C/min (rapid freezing rate). Heterospermic semen samples from chicken breeds raised as part of a Spanish genetic resource conservation program were used in all assays. The 1-min equilibration treatment was associated with a lower percentage of viable thawed spermatozoa than the 30-min treatment (P < 0.05). The remaining sperm variables studied were not affected by equilibration time. The medium-rate 2-step freezing method was associated with a higher percentage of motile spermatozoa after thawing and with greater acrosome integrity (P < 0.05) than the slow nitrogen vapor or rapid 1-step methods. Thawed sperm movement quality and plasma membrane integrity (as assessed by the hypoosmotic swelling test) were better (P < 0.05) in samples frozen by the medium-rate 2-step freezing method than in those subjected to the slow nitrogen vapor method. Fertility was not influenced by freezing method, although that achieved with the medium rate 2-step freezing method showed a trend toward being greater than that achieved with the rapid 1-step method (P = 0.07). Together, the present results suggest that slow cooling rates are not recommendable when using dimethylacetamide. The 2-step freezing method may be useful in the establishment of a germplasm bank for Spanish chicken breeds.

Effects of extracellular environment factors on the motility in Japanese eel spermatozoa

We have examined the effects in vitro of the ionic composition, pH and temperature on the motility by the spermatozoa of the Japanese eel (Anguilla japonica). Milt was obtained from 10 males that had been artificially matured by repeated injections of hCG. Sperm motility was monitored with a VHS video recorder and a video camera connected to a microscope. The results showed that most of the sperm were highly motile in 250-700 mM NaCl, 250-650 mM KCl and 350-550 mM CaCl2 solution. The longest duration of sperm motility recorded in 500 mM NaCl, 250 mM KCl and 350 mM CaCl2 solution. Sperm was not motile when suspended at pH 2, sperm motility was observed at pH 3, there was a relatively higher percentage of motile sperm in solutions at pH 4-12 (above 80%). The motility and duration increased within 18-24°C and decreased at the range of 24-30°C. Appropriate K+ ion concentration in the active medium could enhance the percent motility and duration of eel spermatozoa.

Use of Cholesterol in Sperm Cryopreservation: Present Moment and Perspectives to Future

Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

103 CHOLESTEROL-LOADED CYCLODEXTRIN ADDED TO FRESH BOAR EJACULATES AND SPERM CRYOSURVIVAL

Sperm cryosurvival is affected by altering the lipid composition of sperm plasma membranes and causes damage to spermatozoa during the cryopreservation process as loss of motile cells and functionality, compared with fresh sperm. Our objective was to compare the effect of adding cholesterol-loaded cyclodextrin (CLC) on sperm quality after freezing boar sperm. The CLC was prepared as described: 200 mg of cholesterol was dissolved in 1 mL of chloroform, and 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass dish and the solvents removed using a hot plate for 24 h. The crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL of BTS at 37°C. Thirty-five ejaculates from 5 boars were collected, diluted 1:1 in Beltsville thawing solution, and kept to 2 h at 22°C. The ejaculates were held at 15°C for 60 min and centrifuged at 15°C for 400g for 10 min; the pellet was suspended to 120 million cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk) and divided in 2 treatments: control and 1.5 mg of CLC/mL. The samples were incubated for 15 min at 15°C, cooled to 5°C over a 90-min period, and diluted 1:1 with freeze diluent (72.5 mL of lactose solution 11%, 6 mL of glycerol, 1.5 mL of Equex). Sperm were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen. Straws were thawed in a water bath at 37°C for 30 sec, extended in Beltsville thawing solution, and analyzed by optic microscopy. Sperm were stained with 35 μg mL-1 of Hoechst 33342 and incubated for 15 min at 37°C, centrifuged at 400g for 5 min, and suspended in BTALP to a final concentration of 2 million spermatozoa/mL. A total of 10 000 spermatozoa (5 μL) from each sample were added to droplets containing 10 porcine oocytes. Porcine cumulus oocyte complexes were aspirated and placed in BTALP. The cumulus cells of the oocytes were removed by vortexing for 2 min at maximum speed. Denuded oocytes were washed 4 times in BTALP and incubated for 1 h at 38.5°C in an atmosphere of 5% CO2 in air, following which 10 oocytes per treatment were randomly placed into 45 μL droplets of BTALP, using a small bore fire polished glass pipette to remove loosely bound spermatozoa. Five oocytes were placed onto glass slides and covered with a cover slip supported by a mix of paraffin wax and petroleum jelly. Oocytes were viewed using an epifluorescence microscope, and the total number of spermatozoa bound to each zona pellucida (ZP) was determined at 400× magnification. Treatment differences for sperm motility and zona binding were determined using ANOVA. The addition of CLC to boar sperm before cryopreservation resulted in higher percentages of motile sperm and higher numbers bound to the ZP (35% and 67 sperm/ZP) compared with control cells (26% and 36 sperm/ZP; P &lt; 0.01). In summary, adding CLC to boar sperm before cryopreservation improved cells. FAPEMIG, Piglandia, CNPq, FACEPE.