Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen.

The influence oftemperature, addition of glycerol, initial freezing temperature, method of dilution, level of glycerol in the diluted semen, equilibration time and type of diluent on the survival and fertilizing capacity of deep-frozen according to the best conditions was compared with that of "fresh" semen. The addition of glycerol at plus30 degrees C resulted in a highly significant decrease in the mean proportion of motile spermatozoa immediately after thawing compared with the effect of addition at plus 4 degrees C. The immersion of the straws at minus55 degrees C significantly reduced the revival of the spermatozoa compared with initial freezing at lower temperatures. The exposure time to glycerol had no significant effect on the survival of spermatozoa after thawing and incubation, but fertility was significantly higher with 4% than with 2% glycerol. The I. N. R. A. diluent provided better sperm survival and a significantly higher conception rate than did lactose-egg yolk extender. The semen frozen according to the best conditions (about 50% of the samples) had a fertilizing ability similar to that of "fresh" semen when the proportion of motile spermatozoa before, and after 1 or 3 hr of incubation was equal to or above 45, 40 and 30% respectively.     

Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol

When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to -70 degrees C for solid CO2 and -70 to -196 degrees C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22.4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen-thawed ICR spermatozoa was significantly improved (35.5%) by addition of glycerol (1.75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1.75% glycerol, although the fertilization rates of frozen-thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen-thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.     

Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro.

This paper describes the effects of whole seminal plasma and of dialysed seminal plasma on the fertilizing ability of fowl spermatozoa stored for 24 h at 4 degrees C. The fertilizing ability of fowl semen diluted 1:1 with Beltsville Poultry Semen Extender and stored for 24 h at 4 degrees C was enhanced after replacement of the homologous seminal plasma by the diluent (89 versus 77% fertilization rate). Better results were obtained with seminal plasma dialysed against water before sperm storage to discard the less than 1 kDa or the less than 50 kDa fractions. It was concluded that low molecular weight seminal plasma fractions could damage the fertilizing ability of spermatozoa during storage at 4 degrees C, whereas high molecular weight fractions appeared to enhance fertilizing ability.     

The effects of age on the composition of uterine fluid of broiler breeder hens and on maintenance of quality of fowl spermatozoa when stored in uterine fluid or in a synthetic medium

We studied effects of aging of hens on some physio-chemical parameters of uterine fluid taken at 2 stages of the ovulatory cycle (12 and 18 h after the oviposition) and of uterine fluid properties as a semen diluent in vitro at 41 degrees C. The volume, pH, osmotic pressure, Na+, K+, Ca2+, glucose and total protein concentrations of uterine fluid were measured. The percentage of motile spermatozoa and the fertilizing ability of fowl semen diluted 1:9 in uterine fluid were tested throughout the laying cycle in meat-type hens and compared to those of semen diluted in synthetic medium M199. The results showed that the volume and the K+ and Ca2+ concentrations in uterine fluid decrease with the hens' aging, while osmotic pressure is significantly higher in older hens. But in spite of the composition differences, uterine fluids from hens of different ages have little influence as semen diluents either on sperm motility or on fertilizing ability. These observations suggest that the variations of the composition of uterine fluid with hens' aging does not contribute to the late seasonal decline in fertility observed during the later stages of the reproductive season in this species. In any case, uterine fluid is harmful to sperm quality in vitro, while M199 is an appropriate semen diluent at 41 degrees C. The high glucose concentration in M199, however, slightly decreases motility and the duration of the fertile period of spermatozoa.     

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37 degrees C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-microL drops at 37 degrees C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1 x 10(6)/mL capacitated and motile spermatozoa. A significant difference (P < 0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 +/- 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% CI) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had < 37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 +/- 24.29, while between 2 fertile dogs it was 22.38 +/- 10.02 (P < 0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.     

Rabbit sperm function after co-culture with uterine epithelial cells

The effects of spermatozoa:uterine epithelial cell interactions in vitro on various sperm functions were studied using monolayers of uterine epithelial cells, endometrial stromal cells and fetal fibroblasts. Epithelial and stromal cells were isolated from uteri of rabbits in estrus, while fibroblasts were derived from 12-d-old rabbit fetuses. Twenty-nine to 31 h after culture initiation, washed, ejaculated rabbit spermatozoa were incubated with epithelial cells, uterine stromal or fetal fibroblastic cells, medium or conditioned medium. Sperm viability and loss of acrosome were measured after 10 to 20 h of incubation. Progressive sperm motility and fertilizing ability, which was assessed by an in vivo fertilization assay, were determined after 12 h co-culture. Sperm viability decreased throughout the culture period and was not affected by treatment. Sperm co-cultured with epithelial cells or incubated in medium had fewer acrosomes after 20 h than after 10 h. Fewer sperm co-cultured with stromal or fibroblastic cells lost their acrosomes. Progressive motility was positively affected by sperm-epithelium interaction as 39% of the co-cultured sperm were motile compared with 18% of the sperm incubated in media. In vivo fertilization experiments suggested that sperm incubated with epithelial cells or in medium had similar fertilizing ability. The co-culture of sperm with uterine cells provides an in vitro model to evaluate the effect of gamete-genital tract interaction on sperm function.     

Effects of temperature upon capacitation of guinea pig spermatozoa

Spermatogenesis in many mammalian species requires a temperature a few degrees below body core temperature. Upon ascent through the male tract and deposition in the female tract, the temperature of spermatozoa is increased to body core temperature. This report investigates the effects of temperatures above or below normal body core temperature, which is also the usual temperature of in vitro gamete incubations and fertilization, upon sperm acrosome reacting ability and fertility. Epididymal guinea pig spermatozoa were preincubated in a Ca2+-free medium at temperatures of 15 degrees C, 25 degrees C, 37 degrees C, or 44 degrees C for increasing periods of time. At 15 degrees C or 25 degrees C, no or very few spermatozoa acquired the ability to acrosome react upon exposure to Ca2+ even after 18 hr of culture or warming up to 37 degrees C. A known stimulator of acrosome-reacting ability, lysophosphatidylcholine, was ineffective in promoting acrosome-reacting ability in spermatozoa incubated at 15 degrees C or 25 degrees C. At 37 degrees C the percentage of acrosome reaction increased steadily over time, reaching about 65% after 18 hr. At 44 degrees C the time course of acquisition of acrosome-reacting ability was greatly accelerated with a percentage at 2 hr comparable to that achieved at 37 degrees C only after 18 hr of preincubation. This effect of incubation at 44 degrees C could be reversed by cooling the spermatozoa to 37 degrees C before they were exposed to Ca2+. Spermatozoa induced to undergo the acrosome reaction after preincubation at 44 degrees C were fully capable of fertilizing intact guinea pig eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P < 0.05), with 40% +/- 4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean +/- SEM of 74% +/- 5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52% +/- 8 (P < 0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean +/- SEM of 1.25 +/-.09 for swim-up separated spermatozoa and 1.14 +/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P < 0.05), but there was no effect of separation procedure on development (Day 7) to the morula + blastocyst or blastocyst stage (P>0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.     

Effect of bovine serum albumin on motility and fecundity of turkey spermatozoa before and after storage.

Motility characteristics of turkey spermatozoa before and after storage for 24 h at 7 degrees C in diluent with and without bovine serum albumin (BSA; 1% final concentration) were measured by computer-assisted semen analysis. BSA significantly increased the percentage of motile spermatozoa and sperm velocity, linearity, lateral head displacement and beat frequency in each treatment, but BSA in fresh or stored semen in diluent did not augment hen fertility over 15 weeks of egg production. Fatty-acid-free BSA, globulin-free BSA and Fraction V BSA all significantly increased each sperm motility characteristic compared with semen in diluent alone. The lack of correlation between sperm motility and fecundity emphasizes the need to develop procedures for semen evaluation that accurately predict the fertilizing capacity of an aliquot of semen.     

La Cryoconservation de la laitance de la carpe, Cyprinus carpio

Cryo-preservation of carp, Cyprinus carpio, sperm Deep-freezing trials of carp sperm were carried out by varying several factors such as the basic saline solution, the cryoprotectors added (glycerol, propanediol, ethylene glycol and DMSO), the media (Menezo-INRA B², egg yolk, urea) and the deep-freezing and dilution rates. The success of deepfreezing was judged by the percentage of motile spermatozoa, intensity of motility, fertilizing ability and morphological integrity of the spermatozoa studied under the scanning electron microscope. DMSO was the best cryoprotector and the mineral composition of the dilution medium the least important factor, but there was noticeable improvement after organic compounds were added. The following mixture has been proposed: NaCl 100 mM + KC1100 mM, Tris 20 mM, pH 8: 37%, Menezo medium B² INRA: 15%, urea 5%, DMSO: 10%, fresh sperm: 33%. Optimal deep-freezing rate was: 5°C/min from 2 to-7°C and 25°C/min from-7 to-70°C. In these conditions, about 70 to 80% of the spermatozoa were motile after thawing compared to fresh control sperm, but fertilizing ability was not more than 30 to 40% of that with fresh sperm. The percentage of spermatozoa considered intact was 66% after thawing as against 83% for fresh control sperm. The motility and fertilizing ability of deep-frozen sperm were significantly improved when the dilution rate at insemination was reduced from ¹/₁₀₀ to 1/2.     

Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen

The use of chilled-stored stallion semen is limited by its relatively short-term fertilizing capacity. An important reason for the decrease in fertility during storage is the peroxidation of sperm membrane lipids. In this study, effects of the antioxidants ascorbic acid (0.45 and 0.9 g/L) and catalase (0.45 x 10(6) and 1.8 x 10(6) units/L) on chilled-stored stallion semen were investigated. Semen was collected by artificial vagina from 7 stallions and was diluted with skim milk extender or glycin extender. Sperm motility and membrane integrity were investigated after dilution and after 24, 48 and 72 h at 5 degrees C. Ascorbic acid significantly increased the percentage of membrane-intact spermatozoa at 24, 48 and 72 h at 5 degrees C when compared with that of the controls (P < 0.05), irrespective of the extender. Ascorbic acid decreased the percentage of progressively motile spermatozoa (P < 0.05) at a concentration of 0.9 g/L in glycin extender. Catalase decreased (P < 0.05) progressively motile spermatozoa after 24, 48 and 72 h at 5 degrees C in skim milk extender at a concentration of 1.8 x 10(6) units/L. Catalase decreased (P < 0.05) the percentage of membrane-intact spermatozoa at 24 h. Motility and membrane integrity of spermatozoa after dilution with glycin extender containing catalase did not differ from the controls. In conclusion, ascorbic acid has protective effects on sperm membrane integrity in diluted stallion semen.     

Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing. We hypothesized that freezing/thawing impairs sperm metabolism and that swim-up, but not density gradient centrifugation, recovers metabolically-normal spermatozoa. Ejaculates were cryopreserved, thawed, and processed by swim-up, Accudenz gradient centrifugation, or conventional washing (representing the 'control'). Sperm glucose and pyruvate uptake, lactate production, motility, and acrosomal integrity were assessed. Mitochondrial membrane potential (MMP) was measured in cat spermatozoa. In both species, lactate production, motility, and acrosomal integrity were reduced in post-thaw, washed samples compared to freshly-collected ejaculates. Glucose uptake was minimal pre- and post-cryopreservation, whereas pyruvate uptake was similar between treatments due to high coefficients of variation. In the cat, swim-up, but not Accudenz processing, recovered spermatozoa with increased lactate production, pyruvate uptake, and motility compared to controls. Although confounded by differences in non-specific fluorescence among processing methods, MMP values within treatments were positively correlated to sperm motility and acrosomal integrity. Cheetah spermatozoa isolated by either selection method exhibited improved motility and/or acrosomal integrity, but remained metabolically compromised. Collectively, findings revealed a metabolically-robust subpopulation of cryopreserved cat, but not cheetah, spermatozoa, recovered by selecting for motility rather than morphology.     

Improving the fertilizing ability of sex sorted boar spermatozoa

The sex sorting of spermatozoa by flow cytometry induces damage, since sperm cells are highly diluted, affecting their functionality and fertilizing ability. In this work it was investigated whether the concentration of sex sorted spermatozoa by the sedimentation method, rather than centrifugation, in combination with the presence of the seminal plasma protein PSP-I/PSP-II heterodimer may improve their fertilizing ability. Spermatozoa were sorted by flow cytometry and collected in BTS with 10% of seminal plasma (group C: control) or with 1.5mg/mL of PSP-I/PSP-II heterodimer (group H). Collected spermatozoa from each medium were split into two aliquots. One aliquot of each group was centrifuged (800 x g/5 min) just after sorting and stored 16-18 h at 17 degrees C (groups Cc and Hc) at 6 x 10(6)sperm/mL. The second aliquot was directly stored at 17 degrees C for 16-18 degrees C (group Cs and Hs). After storage the supernatant was discarded and the sedimented pellet adjusted to 6 x 10(6)sperm/mL. Membrane integrity, acrosome status and motility characteristics of spermatozoa from all groups were assessed. Post-weaning pre-ovulatory sows were inseminated by laparoscopy into the oviduct with 0.3 x 10(6) sex sorted spermatozoa to assess their ability to penetrate oocytes in vivo. Putative zygotes were collected 18 h after insemination by washing the oviduct. Penetration and monospermic rates were evaluated. After 16-18 h of storage, centrifuged spermatozoa collected with 10% seminal plasma or 1.5 mg/mL PSP-I/PSP-II heterodimer after sex sorting showed lower (p<0.05) percentages of membrane integrity, motility and fertilization than sedimented spermatozoa. Overall, the presence of 10% seminal plasma or PSP-I/PSP-II heterodimer did not affect the results. However, a positive effect of PSP-I/PSP-II heterodimer (p<0.05) was observed in sedimented spermatozoa. Hence, our results indicate that the sedimentation method in the presence of PSP-I/PSP-II heterodimer improves the in vivo fertilizing ability of sex sorted boar spermatozoa.     

Zinc does not inhibit the capacitation of human spermatozoa in vitro

The effects of zinc on human sperm motility, fertilizing capacity (as assessed by penetration of human spermatozoa into the zona pellucida-free hamster oocyte), and nuclear chromatin decondensation were investigated using spermatozoa from four fertile donors. Both sperm motility and the penetration of sperm into zona-free hamster ova were consistently impaired in media containing 1,000 μM zinc. Spermatozoa from one man were similarly affected at a concentration of 500 μM zinc, but no adverse effects were noted at this zinc concentration in experiments with other donors. Since decreased fertilizing capacity in response to zinc was always accompanied by a significant decline in both the percentage of motile cells and mean swimming speeds, it appears that all of these results reflect a general toxic effect on the cells. At lower concentrations (125–250 μM), zinc had no effect on human sperm motility nor their ability to undergo capacitation and penetrate zona-free hamster ova in vitro. For some donors, zinc (125–500 μM) stimulated both the attachment of spermatozoa to the hamster vitellus and the incorporation of spermatozoa into the hamster ooplasm. The decondensation of human sperm nuclear chromatin in sodium dodecyl sulfate was largely inhibited when zinc was added to the medium, but no significant changes in nuclear stability were apparent after capacitation in zinc-free medium. We conclude that zinc, when present in subtoxic concentrations, does not adversely affect the ability of human spermatozoa to undergo capacitation and penetrate zona-free hamster ova in vitro.     

Separation of motile spermatozoa from frozen-thawed buffalo semen: swim-up vs filtration procedures

Three experiments were conducted to maximize the recovery rate of motile spermatozoa from frozen-thawed buffalo semen. In Experiment 1, the swim-up of motile spermatozoa was performed in the presence or absence of HEPES in TALP medium and CO2 in the environment. The recovery rate of motile spermatozoa in TALP medium (control), TALP + HEPES + CO2, TALP + HEPES and TALP + CO2 was 15, 18, 12 and 10%, respectively (P > 0.05), with sperm motility at 87, 89, 90 and 90%, respectively (P > 0.05). In Experiment 2, the pH of TALP medium was adjusted to 7.0, 7.5, 8.0, 8.5 and 9.0, and swim-up procedure was performed in the presence of HEPES and CO2. The recovery rate of motile spermatozoa at different pH was 14, 20, 24, 27 and 16%, respectively (P < 0.05). Motility of separated spermatozoa was 88, 91, 90, 89 and 90%, respectively (P > 0.05). In Experiment 3, the efficiency of ion-exchange filtration and Swim-up procedure in separating motile spermatozoa from frozen-thawed buffalo semen was compared. The recovery rate of motile spermatozoa was 95% in filtration procedure and 33% in swim-up procedure (P < 0.005). In all experiments, normal acrosomes did not vary due to treatments (P > 0.05). In conclusion, HEPES and CO2 had no significant effect on swim-up of buffalo spermatozoa. The pH 8.5 of TALP improved the recovery rate of motile spermatozoa in swim-up procedure. The ion-exchange filtration was found superior to swim-up procedure in harvesting maximum number of motile spermatozoa from frozen-thawed buffalo semen (95 vs 33%; P < 0.001).     

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

Percoll gradient-centrifuged capacitated mouse sperm have increased fertilizing ability and higher contents of sulfogalactosylglycerolipid and docosahexaenoic acid-containing phosphatidylcholine compared to washed capacitated mouse sperm

Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.     

Relative efficacy of conventional sperm parameters and sperm penetration bioassay to assess bull fertility in vitro

Frozen semen samples from 10 bulls were thawed and actively motile sperm recovered using a swim-up technique. Calcium ionophore A23187 at 0.5 microM concentration was used for 1 min to induce the acrosome reaction in the sperm. Mature female golden hamsters were superovulated with 50 IU of equine chorionic gonadotrophin followed 56 h later with 75 IU of human chorionic gonadotrophin. The cumulus mass was recovered 17 h after hCG treatment by puncturing the oviducts in the infundibulum region. Subsequently, cumulus cell mass and zona pellucida were digested by 0.1% hyaluronidase and 0.1% trypsin, respectively, to yield zona-free hamster eggs (ZFE). A sperm penetration bioassay was performed by coincubating capacitated sperm at 5 X 10(6) concentration and ZFE for 3 h at 38 degrees C in an air incubator. The conception rate of the bulls was based of an average of 82.6 cows per bull with pregnancy status confirmed by rectal palpation. It was found to be strongly correlated (p < 0.01, r = 0.723) with fertilization percentage, whereas percent motile sperm, percent viable sperm and percent sperm with intact acrosomes were not significantly correlated with the conception rate (r = 0.210, -0.021 and -0.468, respectively). Results of the present study suggest that the sperm penetration bioassay can be reliably used to test the fertilizing potential of bull sperm in vitro.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

Quantitative characteristics of Atlantic halibut, Hippoglossus hippoglossus L., semen throughout the reproductive season

The overall objective of the study was to investigate changes in quantitative parameters of Atlantic halibut (Hippoglossus hippoglossus L.) semen throughout the reproductive season in order to systematize the knowledge about biology of Atlantic halibut spermatozoa. Semen samples were collected from February to May from broodstock males kept under either a natural or 3-month advanced photoperiod regime. Spermatozoa concentration, semen pH and osmolality, as well as spermatozoa motility parameters were investigated. The use of catheterization of sperm was examined. Also, fertilization tests were performed. We found that spermatozoa concentration increases in a linear-like mode towards the end of the spawning season, which correlated with a decrease in a number of spermatozoa motility parameters, including actual percentage of motile spermatozoa (MOT), curvilinear velocity (VCL) and straight-line velocity (VSL) of spermatozoa. A breakpoint in MOT occurred when spermatozoa reached a concentration in the range of 17-20 x 10(9) spermatozoa/mL. The fertilization ability of sperm from males kept under natural photoperiod decreased in April. Survival of embryos at 80 degrees days produced by fertilizing eggs of single female with sperm from natural photoperiod males was 88, 76 and 41% on April 09 and 17, and May 01, respectively, whereas using sperm from 3-month delayed photoperiod males for fertilizing eggs from the same female on April 27 resulted in 80% of surviving embryos, not differing significantly from the data from April 09. Physical decomposition of spermatozoa was observed towards the end of the season and it was related to an increase in the whole semen osmolality. Catheterization of semen did not improve spermatozoa motility parameters, however, it reduced the variation in recorded values, especially in the case of pH, caused by contamination with feces or urine. Post-seasonal decrease in spermatozoa concentration was likely related to intensive ageing processes. Based on the present study and available data by other researchers, a model of changes of quantitative parameters in Atlantic halibut semen throughout the reproductive season is proposed.