Assessment of the membrane integrity of fresh and stored turkey spermatozoa using a combination of hypo-osmotic stress fluorescent staining and flow cytometry

A study was conducted to evaluate the integrity of turkey sperm plasma membrane subjected to various hypo-osmotic conditions, and to develop a test to determine the percentage of viable spermatozoa capable of withstanding hypo-osmotic stress after in vitro storage. Semen from 10 toms was collected and pooled twice weekly for 6 wk, and each trial was repeated 6 times. For Trial I, spermatozoa were subjected to varying osmotic solutions by suspension in 100, 80, 60, 40, 20 or 0% PBS in distilled water (297 to 19 mosm/kg H₂O) and stained to assess membrane integrity with Calcein-AM (CAL) and propidium iodide (PI). The CAL detected viable spermatozoa (green fluorescence) while the PI stained dead cells (red fluorescence). Spermatozoa were evaluated microscopically and by flow cytometry. The percentage of viable spermatozoa, as determined by flow cytometry, was not different from that in 100% PBS (76.4 ± 3.8) to 20% PBS (74.1 ± 3.5). Fewer viable spermatozoa, however, were detected in 0% PBS (61.1 ± 4.8, P < 0.05). The percentages of swollen tails observed for viable (green stained) spermatozoa were 0, 4.5, 6.5, 24.3, 50.5 and 100% for 100, 80, 60, 40, 20 and 0% PBS, respectively. Semen was also evaluated fresh or after 24 h in vitro storage at 5 °C in PBS or H₂O (Trial II). The percentage of viable spermatozoa was not different for fresh or in vitro-stored spermatozoa in PBS. For spermatozoa stored 24 h in vitro, the percentage of viable cells was lower in H₂O (48.0 ± 5.1) than in PBS (66.1 ± 5.6, P < 0.05). Subjecting in vitro-stored sperm cells to hypo-osmotic stress before fluorescent staining resulted in detection of labile spermatozoa not accounted for by staining alone, indicating that the turkey sperm membrane is more susceptible to damage after cold storage.     

Phospholipase A2 activation by hydrogen peroxide during in vitro capacitation of buffalo spermatozoa

Progressively motile, washed buffalo spermatozoa (50 x 10(6) cells in 0.5 ml) were in vitro capacitated in HEPES containing Bovine Gamete Medium 3 (BGM3) in presence of heparin (10 microg/ml), and different concentrations of hydrogen peroxide (10 to 100 microM). Spermatozoa (60%) were capacitated in presence of heparin compared to 56% in presence of 25 microM H2O2 (optimally found suitable for capacitation). The extent of capacitation was measured in terms of acrosome reaction (AR) induced by lysophosphatidyl choline (100 microg/ml). The acrosome reacted cells were counted after triple staining. Catalase (100 microg/ml) significantly reduced the sperm capacitation to 16-18% when added with H2O2, or alone in the capacitation medium. Phospholipase A2 activity of spermatozoa increased linearly up to 50 microM H2O2 concentration included in the assay system. Moreover, significant increase in phospholipase A2 activity was observed after capacitation by both, the heparin and 25 microM H2O2. The activity was always higher in acrosome reacted cells.     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium

Low-level production of the superoxide anion (O2*-) is an important signal transduction event in sperm function including capacitation; however, excessive production of O2*- can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O2*- in equine spermatozoa. Ejaculated spermatozoa were separated by centrifugation over a Percoll gradient (40:80), and loaded with DHE (2.0 microM) as well as with calcein-acetoxymethylester (CAM, 7.8 nM) to determine cell viability. In Experiment 1, cells were incubated with the xanthine-xanthine oxidase (X, 0.1 mM; XO, 0.01 U/mL) generating system for the production of O2*-, with or without the addition of superoxide dismutase (SOD, 150 U/mL) or the SOD mimetic, Tiron (0.1, 1.0 or 5.0 mM) for 1h. Changes in fluorescence of DHE were determined for the live cell population (calcein-positive cells) by flow cytometry. The DHE fluorescence increased with the X-XO incubation; this increase was inhibited by SOD or Tiron, indicating that DHE is specific for O2*- detection. In Experiment 2, spermatozoa were loaded with DHE/CAM, treated with calcium ionophore A23187 (0, 0.8, or 8.0 microM), and incubated for 15 min. Cell fluorescence was again determined by flow cytometry. Calcium ionophore A23187 increased O2*- production in a dose-dependent manner. In Experiment 3, cells were loaded with DHE/CAM, treated with NADPH (0.0, 0.25, 0.5, or 1 mM) with or without 0.5% Triton X-100, and incubated for 15 min prior to flow cytometry. Cells treated with NADPH with or without 0.5% Triton X-100 did not have O2*- levels that were significantly different from the control. In Experiment 4, spermatozoa loaded with DHE/CAM were incubated under capacitating conditions (1.2 mM dibutryl-cAMP+1.0 mM caffeine) or in control media for 3h. Although O2*- generation increased over time in control and capacitated treatments, spermatozoa incubated under capacitating conditions had higher O2*- production than those incubated in control media. Therefore, DHE was a useful probe for the detection of O2*- in equine spermatozoa and elevation in intracellular calcium as well as capacitation in vitro were associated with increased generation of O2*-.     

Androcoll-E large selects a subset of live stallion spermatozoa capable of producing ROS

The aim of this study was to elucidate if SLC after 24 h storage selects the subpopulation of spermatozoa that better withstands osmotic shock. To test this hypothesis, viability, mitochondrial membrane potential (MMP) and superoxide anion (O(2)(·-)) production of uncentrifuged (UC) and single layer centrifugation (SLC) - selected spermatozoa were analyzed following SLC after storage of the semen. An aliquot of the extended ejaculate (100×10(6) spermatozoa/mL) was centrifuged through a single layer of a silane-coated silica based colloid formulation optimized for equine spermatozoa (Androcoll-E large, SLU, Sweden) and the rest was used as control. UC and SLC-sperm samples were subjected to osmotic challenges (75 and 900 mOsm) with a subsequent return to isosmolarity (300 mOsm) using Biggers-Whitten-Whittingham (BWW) medium. Viability and MMP decreased after the different osmotic stress in UC and SLC spermatozoa, and return to isosmolarity did not reverse these effects. O(2)(·-) production was enhanced after SLC in all osmolarities tested. Interestingly, the percentage of living spermatozoa showing O(2)(·-) production was increased after 900 mOsm stress in UC spermatozoa, this increase being more evident in SLC spermatozoa. Returning spermatozoa to 300 mOsm enhanced this percentage in UC viable cells but not in SLC spermatozoa. The scenario observed for UC spermatozoa shows that O(2)(·-) is produced in response to isolated hyperosmolarities and subsequent osmotic excursions. As the viability, MMP and cell volume remained the same between SLC and UC spermatozoa, we conclude that Androcoll-E large is likely selecting a higher percentage of physiologically O(2)(·-) producing spermatozoa.     

Production of superoxide anion and hydrogen peroxide by capacitating buffalo (Bubalus bubalis) spermatozoa

In the present study attempts were made to detect and quantify the generation of superoxide anion (O(2)(*-)) and hydrogen peroxide (H(2)O(2)) by capacitating buffalo spermatozoa. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50x10(6)mL(-1) and incubated at 38.5 degrees C with 5% CO(2) in air in the absence or presence of heparin (a capacitation inducer) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) or diphenyleneiodonium (DPI, a flavoprotein inhibitor) for 6h. Production rate of O(2)(*-) and H(2)O(2) by spermatozoa at different hours of capacitation were measured by cytochrome c reduction and phenol red oxidation assays, respectively. Spermatozoa generated both O(2)(*-) and H(2)O(2) spontaneously and following stimulation with heparin and a significant increase of O(2)(*-) production was observed in the presence of NADPH. However, DPI inhibited this NADPH-induced O(2)(*-) production and suggested for existence of putative NADPH-oxidase that constitute a specific O(2)(*-) generating systems in buffalo spermatozoa. Results of our study indicated that buffalo spermatozoa generate O(2)(*-) and H(2)O(2) and production of these free radicals is induced during capacitation.     

Effects of caffeine,Ca++, capacitation time,and strain on in vitro fertilization in mice

Oocytes (N=2922) were collected from superovulated female C57B16/J X DBA2/J (B6D2F₁) mice and distributed among 48 treatments consisting of a 2×3×2×2×2 factorial design. The factors were strain of spermatozoa, B6D2F₁ or SJL/J; caffeine concentration in the fertilization medium, 0,2, or 6 mM; time oocytes were exposed to sperm, 1 or 2 hours; Ca⁺⁺ concentration in the capacitation medium, 0 or 1.8 mM; and capacitation time, 1 or 2 hr. Ova were observed 400 min after they were initially exposed to 10⁵ spermatozoa per ml. Ova with two or more pronuclei and a second polar body were considered fertilized, In vitro embryonic development was monitored for 5 days. B6D2F₁ spermatozoa resulted in consistently higher rates of fertilization than SJL spermatozoa, 77.5% vs 38.7% when averaged over other treatments. Caffeine concentrations of 0,2, and 6 mM resulted in respective mean fertilization rates of 50.1%, 58.8%, and 65.4% (P<0.005) when averaged over other factors. Fertilization rates of ova exposed 1 and 2 hr to sperm were 53.0% and 63.3% (P<0.005). B6D2F₁ spermatozoa capacitated in medium with 1.8 mM Ca⁺⁺ fertilized more ova (P<0.01), 83.1%, than when no Ca⁺⁺ was present, 71.9%; this effect was absent with SJL spermatozoa. The effect of capacitation time depended on strain. Fertilization rates with B6D2F₁ spermatozoa were higher, 80.1%, with a 2-hour capacitation time than with a 1-hour capacitation time, 75.0%. Exactly the opposite was true for the SJL spermatozoa; 43.4% for the 1-hour and 34.1% for 2-hour capacitation (P<.01). Development to the blastocyst stage was significantly greater (P<0.025) for ova fertilized by B6D2F₁ (26.8%) than by SJL spermatozoa (17.7%).     

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes

The effect of storage procedure at 5°C on the quality of ram spermatozoa from the cauda epididymis was analyzed. Two strategies were tested at 0, 24, 48 and 72h post-mortem: (1) spermatozoa held in the epididymal fluid and stored either in the cauda epididymis (In-EPID) or in vitro (Ex-EPID), (2) epididymal spermatozoa extended in three media at 320, 370 and 420 mOsm/kg (D320, D370, D420). Analyzed parameters were: osmolality, pH, motility, acrosomal status and viability. In experiment 1, osmolality of the In-EPID samples, but not in Ex-EPID, increased with post-mortem time. Motility of In-EPID spermatozoa in samples, after 24h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72h. In experiment 2, total (TM) and progressive motility (PM) were not significantly affected by storage time for D320 and In-EPID samples. However, the motility of D370 and D420 samples significantly decreased with time. TM and PM of D320 were significantly higher than D370 and D420 at 72h. At 24h, sperm viability was higher for In-EPID (80.7±3.4%) than for the extended samples (44.8±2.9%, 37.7±3.9% and 48.6±6.0% for D320, D370 and D420, respectively), which also decreased faster with time. At 24h, the percentage of damaged acrosomes was low and similar for the four methods of storage, but damaged acrosomes increased with time for D320 and D370. Storing the spermatozoa in the epididymis is a good strategy for maintaining sperm quality in ram, at least for 48h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.     

Redox control of changes in protein sulfhydryl levels during human sperm capacitation

Capacitation, the series of transformations that spermatozoa undergo to become fertile, is regulated by reactive oxygen species (ROS) and associated with an increase in the sulfhydryl content of Triton-soluble proteins. Our aims were to determine the fate of sulfhydryl groups in Triton-soluble proteins from capacitating human spermatozoa using two-dimensional (2D) gel electrophoresis, to evaluate the role of ROS in the changes observed, and to correlate the time course of the changes with that of the sperm generation of O(2)(*)(-). Triton-soluble proteins of control and capacitating human spermatozoa were labeled with 3-(N-maleimidylpropionyl) biocytin, separated by 2D gel electrophoresis, and probed with horseradish peroxidase-conjugated streptavidin. The sulfhydryl content of 10 out of the 14 proteins studied (pI: 4-7) was modified by the induction of capacitation, and the increases (by 200-400%, five proteins) and decreases (by 45-95%, five proteins) were prevented by superoxide dismutase and/or catalase. The alterations in protein sulfhydryl content occurred within 5-15 min but were reversed within 30-120 min. Three capacitation inducers triggered similar modifications. Therefore, human sperm capacitation is associated with rapid and reversible changes in protein sulfhydryl groups that appear to be redox regulated. The number of proteins affected, the types, and the kinetics of changes emphasize the complexity of sperm capacitation.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. IV. Modification of genetic damage induced by X-irradiation of spermatozoa and spermatids in N2 or O2 by mei-9a, mei-41D5 and mus(1)101D1

The influence of defects in DNA repair processes on X-ray-induced genetic damage in post-meiotic male germ cell stages of Drosophila melanogaster was studied using the 'maternal effects approach'. Basc males were irradiated in N2, air or O2 either as 48-h-old pupae (to sample spermatids) or as 3-4-day-old adults (to sample mature spermatozoa) and mated to females of 3 repair-deficient strains (mei-9a: excision-repair-deficient; mei-41D5: post-replication-repair-deficient; mus(1)101D1: post-replication-repair-deficient and impaired in DNA synthesis). Simultaneous controls involving mating of males to repair-proficient females (mei+) were run. The frequencies of sex-linked recessive lethals and of autosomal translocations were determined following standard genetic procedures. The responses elicited in the different crosses with repair-deficient females were compared with those in mei+ crosses. The main findings are the following: with mei-9 females, the frequencies of recessive lethals are higher after irradiation of spermatids in N2, but not after irradiation in air of O2 (relative to those in the mei+ crosses); this result is different from that obtained in earlier work with spermatozoa, in which cell stage, higher yields of recessive lethals were obtained after irradiation of males in either N2 or air; in the mei-9 crosses, there are no significant differences in response (relative to mei+) after irradiation of either spermatozoa or spermatids in O2; the translocation frequencies in the mei-9 crosses are similar to those in the mei+ crosses, irrespective of the treated germ cell stage or the irradiation atmosphere; irradiation of either spermatozoa or spermatids in N2, air or O2 does not result in any differential recovery of recessive lethals in the mei-41 relative to mei+ crosses; irradiation of spermatids in N2 and of spermatozoa in air leads to a higher recovery of translocations in the mei-41 crosses; and after irradiation of spermatids or spermatozoa in any of the gaseous atmospheres, the frequencies of recessive lethals and of translocations are lower in the mus-101 crosses. The differences in responses (between cell stages, in different gaseous atmospheres and with different repair-deficient females) are explained on the basis of both qualitative and quantitative differences in the composition of the initial lesions and the extent to which their repair may be affected by the defects present in the different repair-deficient females. Several discrepancies between expectations based on biochemical results and the genetic results are pointed out.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hyperoxia decreases lung size of amphibian tadpoles without changing GSH-peroxidases or tissue peroxidation

1. During the development of D. pictus larvae (Amphibia) in normoxia, selenium (Se) GSH-Px increased whereas non-Se GSH-Px did not change. 2. Acclimation to 60 or 100% O2 did not change Se GSH-Px or non-Se GSH-Px. 3. Hyperoxia did not change tissue peroxidation (TBA-RS) confirming the good capacity of D. pictus tadpoles for O2-adaptation. 4. Since hyperoxic induction of catalase (CAT) has been previously described in D. pictus tadpoles, it is concluded that CAT is more important than both GSH-Px for the establishment of O2-adaptation. 5. Increases of Se GSH-Px, SOD and CAT, are probably important for adaptation to the change from aquatic to aerial environment during metamorphosis in normoxia. 6. Chronic exposure to 100% O2 enormously reduced the lung size of D. pictus larvae.     

Accumulation of deuterium oxide in body fluids after ingestion of D2O-labeled beverages

A simple low-cost procedure was developed to compare the temporal profiles of deuterium oxide (D2O) accumulation in body fluids after ingestion of D2O-labeled solutions. D2O concentration was measured in plasma and saliva samples taken at various intervals after ingestion of 20 ml of D2O mixed with five solutions differing in carbohydrate and electrolyte concentrations. An infrared spectrometer was used to measure D2O in purified samples obtained after a 48-h incubation period during which the water (D2O and H2O) in the sample was equilibrated with an equal volume of distilled water in a sealed diffusion dish. The procedure yields 100% recoveries of 60-500 ppm D2O with an average precision of 5%. When compared with values for distilled water, D2O accumulation in serial samples of plasma and saliva was slower for ingested solutions containing 40 and 15% glucose and faster for hypotonic saline and a 6% carbohydrate-electrolyte solution. These differences appear to reflect known differences in gastric emptying and intestinal absorption of these beverages. Therefore this technique may provide a useful index of the rate of water uptake from ingested beverages into the body fluids.     

Respiratory burst and NAD(P)H oxidase activity are involved in capacitation of cryopreserved bovine spermatozoa

Heparin (a glycosaminoglycan) and quercetin (a calcium-ATPase plasma membrane specific inhibitor) induce bovine sperm capacitation. Mitochondria from frozen semen are capable of generating oxidative energy. The aim of the study was to determine oxygen uptake variation and the participation of diphenileneiodonium (DPI)-sensitive oxidases from spermatozoa capacitated with heparin or quercetin. Oxygen uptake was measured polarographically and 2 microM diphenileneiodonium (DPI) was used as a specific inhibitor of NAD(P)H-oxidases. Sperm capacitation was determined by the chlorotetracycline technique. Heparin produced a respiratory burst (17.0+/-3.2 microL O2/h/10(8) spermatozoa; mean+/-S.D.) versus control (11.3+/-0.9 microL O2/h/10(8) spermatozoa; P<0.05). Oxygen uptake and sperm hypermotility were inhibited by cyanide. Treatment with DPI blocked heparin capacitation and oxygen uptake (cyanide-sensitive) decreased to control levels. Respiration of quercetin-treated samples (cyanide-sensitive; 9.7+/-0.7 microL O2/h/10(8) spermatozoa) was not significantly different from the controls; oxygen uptake was not modified by DPI, but quercetin capacitation was inhibited (P<0.05). The effect of DPI with heparin confirmed that oxidases participate in capacitation induction. The addition of superoxide dismutase and/or catalase to heparin- or quercetin-treated samples, failed to modify oxygen uptake and blocked capacitation (P<0.05), suggesting that the superoxide anion (O2*-) participates in the capacitation induction. High mitochondrial activity from heparin-treated samples indicated that energy requirements, especially for hypermotility, were supported by the respiratory chain. Although a respiratory burst was not produced by quercetin, DPI-sensitive-oxidases (O2*- source) were necessary for capacitation. In cryopreserved bovine spermatozoa, heparin- or quercetin-induced capacitation required different levels of mitochondrial energy and DPI-sensitive oxidase activity.     

Effects of bull and heparin and sperm concentrations on in vitro fertilization of buffalo (Bubalus bubalis ) oocytes matured in vitro

A study was undertaken to assess the ability of spermatozoa from 6 buffalo bulls, at different levels of heparin and sperm concentrations, to achieve an acceptable level of fertilization in vitro. Frozen-thawed spermatozoa, 3 dosages of heparin (0, 10 and 100 ug/ml) in the presence and absence of penicillamine, hypotaurine and epinephrine (PHE), and 4 sperm concentrations (1 x 10(6), 2 x 10(6), 3 x 10(6) and 4 x 10(6) /ml) were studied using 3202 buffalo oocytes. The mean proportions of fertilized oocytes in the group treated with 10 ug/ml of heparin were significantly higher (P<0.05) with the semen of Bulls A, B and C (44.7 to 64.3%) than in medium devoid of heparin. An increase in the dosage of heparin from 10 ug/ml to 100 ug/ml reduced the overall fertilization rate. However, optimal fertilization (30.9%) at 100 ug/ml heparin was observed for semen from Bull D. Bulls E and F yielded the lowest fertilization rate (9.6 and 14.2%, respectively) at the above mentioned heparin dosage. Analysis of sperm density revealed that a concentration of 2 x 10(6) spermatozoa yielded optimal fertilization rates in vitro. Higher sperm concentrations (3 x 10(6) or 4 x 10(6)) resulted in higher oocyte penetration rates but gave rise to polyspermy.     

Post-thaw survival of ram spermatozoa and fertility after insemination as affected by prefreezing sperm concentration and extender composition

A study was conducted to investigate the effects of prefreezing sperm concentration using two extenders on post-thaw survival and acrosomal status of ram spermatozoa (Experiment 1) and fertility after intrauterine insemination with differing doses of semen (Experiment 2). In autumn (Northern hemisphere), semen was collected by artificial vagina from 8 adult Leccese rams and ejaculates of good quality semen were pooled. Two extender systems for cryopreservation were considered, one based on milk-lactose egg yolk (Milk-LY) and the other based on tris-fructose egg yolk (Tris-FY). Experiment 1 (2 x 6 factorial scheme) examined the in vitro characteristics of spermatozoa in relation to the Milk-LY and Tris-FY extenders and six prefreezing sperm concentrations (50, 100, 200, 400, 500 and 800 x 10(6) spermatozoa/mL). Experiment 2 (2 x 4 factorial) evaluated the influence of the Milk-LY vs Tris-FY extenders and four doses (20, 40, 80 and 160 x 10(6) spermatozoa/0.25 mL) corresponding to prefreezing spermatozoa concentrations of 100, 200, 400 and 800 x 10(6) spermatozoa/mL, on fertility of ewes inseminated in uterus by laparoscope. Prefreezing sperm concentration influenced (P < 0.01) freezability of spermatozoa and affected negatively all the in vitro parameters at 800 x 10(6) spermatozoa/mL. Overall, Milk-LY tended to ensure higher viability and acrosomal integrity of spermatozoa after thawing at the intermediate sperm densities (range 100 to 500 x 10(6) spermatozoa/mL). At 500 x 10(6) spermatozoa/mL concentration corresponded the best condition for survival of spermatozoa (71.2%), acrosome integrity (71.5%) and acrosomal loss (6.0%). At the lowest sperm concentration (50 x 10(6) spermatozoa/mL), Tris-FY resulted in a higher survival rate than Milk-LY (61.3%, P < 0.05) and lower acrosomal loss (9.7%, P < 0.05). Milk-LY supported spermatozoa motility better than Tris-FY after incubation at sperm concentration between 50 and 400 x 10(6) spermatozoa/mL (0.05 > P < 0.01). Semen doses of 20 to 40 x 10(6) spermatozoa/ewe provided satisfactory fertility rates (64 to 81%). The increase of inseminate doses to 160 x 10(6) spermatozoa/ewe failed to improve fertility, actually tending to decrease lambing rates.     

Sperm motility of the marine teleosts Boops boops, Diplodus sargus, Mullus barbatus and Trachurus mediterraneus

The spermatozoa of Boops boops, Diplodus sargus, Mullus barbatus, and Trachurus mediterraneus were motile in sea water, and in electrolyte solutions (NaCl) and non-electrolyte solutions (glucose) with an osmolality of 600–1000 mosmol kg^?1. Their mean motility rate 10 s after initiation was about 80%, while about 10% of the motile spermatozoa moved non-linearly, 45% linearly, and 45% circularly. The average path swimming velocity was significantly higher in M. barbatus (about 90 μm s^?1) than in the other species (70 μm s^?1). The number of motile spermatozoa decreased to 0% within 50 s after initiation of motility in T. mediterraneus, within 90 s in M. barbatus . In B. boops and D. sargus about 90% of the spermatozoa stopped movement during the first 90 s of the motility period, while the rest remained motile for 2–3 h. Motility of B. boops and D. sargus spermatozoa was reversibly suppressed in the seminal plasma, and in electrolyte and non-electrolyte solutions of 100–200 mosmol kg^?1. The trigger for motility activation was hyperosmolality (700–1000 mosmol kg^?1). Motility of M. barbatus and T. mediterraneus sperm was only partly suppressed in the seminal plasma since freshly collected semen contained about 25–50% locally motile spermatozoa. When sperm was activated immediately after collection with electrolyte and non-electrolyte solutions of 700–1000 mosmol kg^?1 spermatozoa moved progressively. The motility of those spermatozoa which had not yet been motile after collection was completely and reversibly suppressed in M. barbatus at osmolalities of 1200 mosmol kg^?1, and at osmolalities of 100–200 mosmol kg^?1 in T. mediterraneus . Therefore two triggers were necessary for initiation of motility. The nature of the first trigger was uncertain, the second trigger was a switch to hypoosmolality in M. barbatus and to hyperosmolality in T. mediterraneus . The sperm organisation of B. boops, D. sargus, M. barbatus and T. mediterraneus revealed species-specific parameters which could not be related with the sperm motility behaviour.     

Effect of time of ovulation on fertilization after intrabursal transfer of spermatozoa (ITS): improvement of a new method for artificial insemination in mice

The timing of AI in relation to ovulation was examined to improve intrabursal transfer of spermatozoa (ITS) in mice, a new method of AI that involves transfer of spermatozoa into a space near the infundibulum. Two microliters of fresh epididymal B6C3F1 spermatozoa (containing 2 x 10(5) spermatozoa) were inseminated 1, 7, 12, or 17 h after hCG administration. At 1.7 days after ITS, normal cleaving embryos were recovered at rates ranging from 6 to 50% (21.5 +/- 15.8%; mean +/- S.D.), 40-100% (75.2 +/- 20.2%), 33-100% (60.1 +/- 19.3%), and 6-47% (22.7 +/- 13.3%), respectively. The rate obtained by ITS 7h after hCG administration was comparable (P > 0.05) to that (90.5 +/- 6.3%) for embryos obtained after natural mating (control), but rates at all other times were significantly less than control. To examine whether in vivo fertilization rate differs when spermatozoa from various mouse strains are used, B6C3F1 females were inseminated with spermatozoa from ICR, C57BL/6N and C3H/HeN mice 7 h after hCG administration. There were strain differences (P < 0.01 for ICR and B6C3F1 versus C57BL/6N and C3H/HeN) for in vivo fertilization rates (83.9 +/- 10.3%, 75.2 +/- 20.2%, 33.6 +/- 24.5% and 25.6 +/- 16.1% for ICR, B6C3F1, C57BL/6N and C3H/HeN, respectively). Similar rates (72.9 +/- 7.3% and 27.5 +/- 46.2% for ICR and C57BL/6N, respectively) were also obtained when oocytes were inseminated with spermatozoa of the same strain. In addition, females (B6C3F1) inseminated by ITS of fresh B6C3F1 spermatozoa 7 h after hCG administration yielded normal mid-gestational fetuses with an average litter size of 7.0 +/- 4.9, which seemed much higher than the previously reported litter size of 3.2. In conclusion, the timing of AI was considered a key factor affecting in vivo fertilization efficiency.     

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.