Sperm Dilution Research Articles

Analysis of motility parameters from paddlefish and shovelnose sturgeon spermatozoa

Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s‐1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s‐1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.

Comparison of protective media and freezing techniques for cryopreservation of human semen

Objective: To evaluate the influence of cryopreservation medium and freezing–thawing techniques on human sperm motility and morphology. Study design: 63 semen samples were obtained from 39 donors to the artificial insemination programme. Possible effects of the sperm dilution with cryomedium on the motility were examined 10 min after exposure of 24 high initial quality semen samples to TEST–yolk {zwitterion–citrate–egg yolk extender containing TES [N-Tris (hydroxymethyl) methylaminoethane sulfonic acid] and Tris [(hydroxymethyl) aminomethane]} and human sperm preservation medium (HSPM). Post-thaw sperm motility from 24 frozen semen samples was examined comparing the cryoprotective efficacy of TEST–yolk and HSPM following different freezing techniques (vapour freezing, fast programmable freezing and slow programmable freezing). The relationship of sperm morphology to the effects of freezing was investigated on 39 semen samples following different freezing techniques. Post-thaw sperm motility from 39 frozen semen samples was compared among three groups divided according to the percentage of morphologically normal cells (<40, 40–50 and >50%) in fresh semen. Results: Exposure of spermatozoa to cryomedia for 10 min at room temperature significantly reduced motility in TEST–yolk treatment group for 9% and in HSPM treatment group for 18% (P<0.01). The recovery of motile sperms (mean±standard deviation) was 49±15.7, 43±15.2 and 52±16.8% when TEST–yolk was used and 34±17.8, 32±18.2 and 50±13.6% when HSPM was used as a cryopreservative following vapour freezing, and fast and slow programmable freezing, respectively. Following vapour freezing and also following fast programmable freezing, the recovery of motile sperm was significantly higher (P<0.05) after addition of TEST–yolk medium than after addition of HSPM. Post-thaw motility of the sperm cryopreserved in HSPM showed significant differences (P<0.05) after three different freezing techniques. The recovery of motile sperms was 57±26.4, 38±8.6 and 38±17.3% in groups with >50, 40–50 and <40% morphologically normal cells, respectively. The percentage of morphologically normal spermatozoa was reduced 8% after vapour freezing and 6 and 3% after fast and slow programmable freezing, respectively. The results were statistically analysed using sas/stat software. Conclusions: Slow programmable freezing was superior to vapour freezing and fast programmable freezing as a method for sperm cryopreservation. However, none of these methods of freezing had discernible effects on sperm morphology. Motility of spermatozoa decreased due to the exposure of semen to cryomedium. TEST–yolk was a superior cryomedium to HSPM. Fresh semen with more than 50% of morphologically normal cells showed the best recovery of motile cells after freezing and thawing.

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar spermatozoa.

Geographic divergence of gamete recognition systems in two species in the sea urchin genus Strongylocentrotus

Two closely related species in the camarodont sea urchin family Strongylocentrotidae, Strongylocentrotus pallidus and Strongylocentrotus droebachiensis, have circumarctic ranges with overlapping depth distributions and spawning seasons. Both species show genetic differences between Europe and the Pacific in the nuclear sperm bindin gene, and S. droebachiensis also differs in mitochondrial DNA. Gamete recognition systems of allopatric populations may or may not have drifted apart, but are unlikely to have diverged by reinforcing selection. Geographic divergence in gamete compatibility may illustrate mutational biases and variability and hence possible initial stages of reproductive isolation.We carried out fertilisation experiments with both species' gametes from Norway and from the Eastern Pacific Ocean. Serially diluted sperm were added to egg suspensions in factorial designs, and fertilisation success was assessed by counting the proportion of cleaving embryos. To separate the specificity of the acrosome reaction from subsequent sperm–egg binding steps, a subsample of sperm was pre-activated by performing the last dilution step in diluted egg jelly from conspecific females. Fertilisation successes were compared by logistic regression ANCOVAs over five, 4-fold sperm dilutions for each cross. The graphs shown here illustrate fertilisation success for one sperm concentration only.

Effect of semen preparation on IVF of prepubertal goat oocytes

The aim of these experiments was to study the effects of different methods of washing and selection of spermatozoa on the IVF of IVM oocytes from prepubertal goats. Fresh ejaculates from 3 males of proven fertility were processed according to the following treatments: 1) centrifugation in TALP, 2) centrifugation in sucrose-based Ficoll medium, 3) centrifugation in Percoll gradients at 40 and 80%, 4) by swim-up and 5) by dilution of spermatozoa (1:40) in (1:1) TALP. In all 5 treatments spermatozoa were incubated for 45 min with 100 μg/mL of heparin and then added to Fert-TALP. Oocytes were matured for 27 h in TCM-199 supplemented with 20% estrous goat serum (EGS), FSH, LH and estradiol-17β. Spermatozoa (4× 10 6 cells/mL) were coincubated with oocytes in 100 μL of Fert-TALP with hypotaurine for 24 h, after which the oocytes were transferred to a granulosa cells monolayer in TCM-199 plus 10% of EGS for 24 h (48 h post insemination). At 17 h post insemination a sample of sperm-exposed oocytes was taken and stained in lacmoid to observe sperm penetration and the formation of pronuclei. At 48 h post insemination the cleavage rate of oocytes was evaluated. Motility, viability and acrosome status of the spermatozoa were evaluated immediately after the mixing of the ejaculates, after washing and selection treatments, and after incubation with heparin and at 17 h post insemination. The different ejaculate treatments did not affect the penetration and cleavage rates of oocytes. At 48 h post insemination the cleavage rate was 46.9, 36.6 and 29.0% for dilution, Ficoll and swim-up preparations, respectively. Only the swim-up protocol improved sperm motility and viability compared with that of the initial semen sample and with the other sample treatments. At 17 h post insemination the semen parameters were the same for all sperm sample treatments.

Gonadal morphology and gametogenesis in the sea pen Pennatula aculeata (Anthozoa: Pennatulacea) from the Gulf of Maine

The ultrastructural features of gametogenesis are described in male and female colonies of the sea pen Pennatula aculeata. Specimens were collected for observation and fixation at 113 to 231 m depth in the Gulf of Maine, USA, in August 1993. The species is gonochoric, and all stages of gametogenesis are observed in both male and female colonies >45 mm in height. Gametogenesis shows several features that differ from sea anemones. The developing oocytes and sperm cysts are completely encompassed by gastrodermally derived follicle cells, and they are released from the mesenteries into the coelenteron before they are fully differentiated. Following maturation in the coelenteron, the eggs and intact sperm cysts are expelled through the mouths of the autozoids during spawning. The expulsion of sperm cysts suggests that they function as primitive spermatophores, perhaps as a way of reducing sperm dilution. Vitellogenesis results in the biosynthesis of lipid droplets which are the sole nutrient reserves in the egg. Heterosynthetic vitellogenesis is characterized by the importation of lipid precursors into the oocyte, and there is some indirect evidence that hypertrophic follicle cells play a role in production, transport, and/or mediation of these precursors. Spermatogenesis is similar to that of other anthozoans. The spermatozoon has a cone-shaped head, a posterior nuclear fossa, a ring of lipid-like bodies in the midpiece, a prominent cytoplasmic collar surrounding the proximal flagellum, and a single mitochondrion, but the posterior region of the sperm also contains previously undescribed concentric rings of cisternae resembling smooth endoplasmic reticulum.

Fertilization in the sea: are the hazards of broadcast spawning avoided when free–spawned sperm fertilize retained eggs?

In broadcast-spawning marine animals, rapid dilution and short lifespan of sperm following release may impose severely localized patterns of mating. Partial or total failure of external fertilization due to sperm limitation appears commonplace. However, it is not clear to what extent the restrictive kinetics of fertilization in water also constrain mating in animals that release sperm but retain their eggs for fertilization. The compound ascidian Diplosoma listerianum liberates sperm that are dispersed to other colonies and taken in prior to internal cross-fertilization. The fertile lifespan of sperm was found to be long (half-life ca. 8 hours), and a substantial number of fertilizations occurred with 24-hour-old sperm. Fertilizations were obtained from sperm concentrations that would typically produce little or no external fertilization. In a separate experiment, a very small piece of D. listerianum (dry weight less than 2 mg) sired abundant progeny throughout a 3840 l tank. Paternity of progeny in these experiments was confirmed by molecular markers. The same markers were used to extend, to over seven weeks, the known maximum period of storage of exogenous sperm prior to fertilization in this species. The production of only a few thousand sperm at a time by each zooid, poor synchronization of release between zooids, and the existence of many well-spaced exhalant openings in large colonies suggest that D. listerianum is incapable of generating a dense plume of sperm, even close to the source. It is suggested that, unlike external fertilization, successful internal cross-fertilization in D. listerianum is not dependent upon the interception of a dense cloud of gametes just released by a near neighbour. It seems instead that dilute, long-lived sperm can be extracted efficiently from seawater by this suspension feeder, potentially over a period of time. This capability, and other features of the life history, make it unlikely that sperm limitation is an acute problem in this species and comparable taxa, a conclusion with potential significance for expected patterns of mating, sex allocation and gamete attributes in sessile aquatic invertebrates. Variance in reproductive success between individuals due to differences in fertilization rate may be much lower than in broadcast spawners exhibiting external fertilization.

Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose

Glycerol is not an effective cryoprotectant for rabbit spermatozoa; therefore, rabbit spermatozoa were used as a model for developing cryopreservation procedures for other cell types which also freeze poorly when glycerol is used as the cryoprotectant. Experiments were conducted to 1) compare several published protocols for cryopreserving rabbit spermatozoa; 2) determine if removal of seminal granules, required for flow cytometry analysis, affects the motility of rabbit spermatozoa; and 3) determine if using a combination of cell permeating cryoprotectants (acetamide) with cell nonpermeating cryoprotectants (trehalose and methyl cellulose; MC), can increase the recovery of viable rabbit spermatozoa after cryopreservation. Media containing acetamide as a cryoprotectant were found to be most effective for rabbit spermatozoa. The cryoprotectants ethylene glycol, dimethylsulfoxide and glycerol were not effective for cryopreserving rabbit spermatozoa. Second, rabbit spermatozoa could be centrifuged through a Percoll gradient composed of equal volumes of Prcoll and a HEPES-buffered sperm medium. This centrifugation removed all seminal granules without affecting the percentage of motile spermatozoa after initial sperm dilution (85 vs 74%) or after cryopreservation (35 vs 30%), when sperm were either centrifuged or not centrifuged, respectively. The substitution of trehalose in the cryopreservation medium for raffinose did not improve recovery of motile cells following cryopreservation (P > 0.05). However, addition of MC resulted in higher percentages of motile sperm after cryopreservation (43 vs 31%; P < 0.05). In addition, sperm viability and acrosomal integrity were simultaneously evaluated using flow cytometry. The addition of both trehalose and MC to media containing acetamide resulted in higher percentages of live acrosome-intact cells than acetamide alone (53 vs 37%; P < 0.05). These results indicate that a combination of permeating and nonpermeating cryoprotectants (acetamide, trehalose and MC) were more effective in preserving rabbit spermatozoa than acetamide alone and that analyzing multiple sperm characteristics, by flow cytometry, can assess sperm damage not detected by analyzing sperm motion characteristics.

The timing of pronuclear formation of IVM/IVF-produced bovine zygotes and their development following DNA microinjection

The aim of these experiments was to study the effects of different methods of washing and selection of spermatozoa on the IVF of IVM oocytes from prepubertal goats. Fresh ejaculates from 3 males of proven fertility were processed according to the following treatments: 1) centrifugation in TALP, 2) centrifugation in sucrose-based Ficoll medium, 3) centrifugation in Percoll gradients at 40 and 80%, 4) by swim-up and 5) by dilution of spermatozoa (1:40) in (1:1) TALP. In all 5 treatments spermatozoa were incubated for 45 min with 100 μg/mL of heparin and then added to Fert-TALP. Oocytes were matured for 27 h in TCM-199 supplemented with 20% estrous goat serum (EGS), FSH, LH and estradiol-17β. Spermatozoa (4× 106 cells/mL) were coincubated with oocytes in 100 μL of Fert-TALP with hypotaurine for 24 h, after which the oocytes were transferred to a granulosa cells monolayer in TCM-199 plus 10% of EGS for 24 h (48 h post insemination).At 17 h post insemination a sample of sperm-exposed oocytes was taken and stained in lacmoid to observe sperm penetration and the formation of pronuclei. At 48 h post insemination the cleavage rate of oocytes was evaluated. Motility, viability and acrosome status of the spermatozoa were evaluated immediately after the mixing of the ejaculates, after washing and selection treatments, and after incubation with heparin and at 17 h post insemination.The different ejaculate treatments did not affect the penetration and cleavage rates of oocytes. At 48 h post insemination the cleavage rate was 46.9, 36.6 and 29.0% for dilution, Ficoll and swim-up preparations, respectively. Only the swim-up protocol improved sperm motility and viability compared with that of the initial semen sample and with the other sample treatments. At 17 h post insemination the semen parameters were the same for all sperm sample treatments.

Progression of meiosis in porcine oocytes matured in vitro and the polyspermy problem

The high incidence of polyspermy is still an unresolved problem for the production of in vitro-produced porcine embryos. In this work, we modified the usual sperm processing sequence for in vitro fertilization (IVF), and the spermatozoa from four boars were frozen directly at a low sperm concentration of 20 × 106 sperm/mL (high pre-freezing sperm dilution group; F20), thawed and processed for IVF in three replicates. Spermatozoa from the same boars frozen at a conventional concentration (1000 × 106 sperm/mL) were used as the control group. The post-thaw sperm quality evaluation demonstrated that despite there being no differences in the percentage of motile spermatozoa between groups, the proportion of live spermatozoa with intact acrosomes was significantly higher in the F20 group than in the control. The in vitro penetration rate was also similar between groups; however, the co-incubation of oocytes with F20 sperm increased monospermy, IVF efficiency, cleavage rate and the efficiency of blastocyst formation compared with the results for oocytes co-incubated with control spermatozoa. These results indicate, for the first time, that a high pre-freezing sperm dilution increases monospermy without affecting penetration rates, thereby increasing blastocyst formation.

Motility of spermatozoa from shovelnose sturgeon and paddlefish

The spermatozoa in the seminal plasma from shovelnose sturgeon Scaphirhynchus platorynchus and paddlefish Polyodon spathula were immotile with only a few spontaneously motile spermatozoa for 5‐10 and 10‐20 s, respectively. Spermatozoa of shovelnose sturgeon were observed to be 100% motile immediately after sperm dilution in 10 mm NaCl and 20 mm Tris‐HCl, pH 8.5. The duration of mass progressive movement was 2‐3 min; and 1 to 5% of spermatozoa remain active after 360 s (P&lt;0.01). Spermatozoa of paddlefish demonstrated the best motility 10 s after dilution in 10 mm NaCl with 20 mm Tris‐HCl, pH 8.5. The duration of mass progressive movement was 2‐3 min and 1 to 5% of spermatozoa remained active after 370 s (p&lt;0.01). The spermatozoa of shovelnose sturgeon and paddlefish were motile in a range of osmotic pressure from 0 to 100 mosmol kg−1 and 0 to 120 mosmol kg−1, respectively. The best results with short‐term storage of sperm from shovelnose sturgeon and paddlefish were observed in 100 mm glucose + 20 mm Tris‐HCl, pH 8.5 and 150 mm glucose + 20 mm Tris‐HCl, pH 8.5.

Sperm motility in turbot, Scophthalmus marimus: initiation of movement and changes with time of swimming characteristics

Turbot sperm motility is observed using dark field microscopy and stroboscopic illumination combined with video recording. Sperm motility is triggered by dilution of spermatozoa in sea water or in non ionic media (glucose or saccharose), presenting osmotic pressure ranging from 300 to 2100 mOsmol. The percentage of motile spermatozoa reaches 100% under conditions of osmotic pressure of 300 to 1100 mOsmol and pH close to 8.0. In full sea water, glucose or saccharose solutions an agglutination of spermatozoa is observed; this is prevented by addition of bovine serum albumin (5 mg ml−1). Immediately after transfer in activation solutions, 100% spermatozoa are motile in most samples freshly stripped. This percentage drops suddenly between 15 and 30% after 70 to 100 sec. The beat frequency remains at a constant value of 50 Hz during 40 s post activation and then drops suddenly between 15 and 30 Hz. The spermatozoa velocity is about 200 micrometers s−1 during 30 to 40 s and then declines to a stable value of 100 micrometers s−1 at 50 s post activation. After 1.20 mn, more and more spermatozoa become motionless. The minimum calculated and averaged distance covered during 1.20 min, is about 12 mm. The high performances of turbot spermatozoa motility are interpreted as a compensatory mechanism for the low sperm production.

Influence du maintien en mer ou de la période de transfert en eau douce des reproducteurs de saumon atlantiqueSalmo salarsur la maturation sexuelle et la qualité des gamètes

Breeding management of Atlantic salmon generally requires transfer of spawners to fresh water pnor to final sexual maturation, in order to avoid poor gamete quality. To study the impact of sea water environment, pre-spawners of Atlantic salmon Salmo salar (2) were either reared in sea cages until final sexual maturation or transferred to fresh water in June or October. The beginning of spermiation or ovulation was recorded by weekly checking using hand-stripping. The quality of spawners was tested by fertilizing batches of 200 eggs, using DIA 532 insemination extender with sperm dilution of and assessing survival at eyeing stage. Males transferred to fresh water (ED) presented normal spermiation cumulative frequency curves in both fresh water groups. Females transferred to fresh water ovulated from November to December while ovulation of females reared in sea water was delayed and the period lengthened. Furthermore, only 67% of the EM females had ovulated during the experiment up to April. Relative fecundity of females transferred to ED in spring was significantly lower than that of females maintained in EM during sumrner. In spite of delayed and lengthened ovulation and of significantly higher osmolality of coelomic plasma (348 I 13 mOsmoVI, in EM and 270 123 mOsmoyl in ED) eggs of EM females were of a similar high quality to those of ED females, when fertilized less that one week after ovulation. Males reared in sea water presented similar but slightly earlier spermiation curves. Heterogeneous and lower mean semen quality were recorded in EM males as compared with the ED males. Semen plasma osmolality was significantly higher in EM males (347 ? 27 mOsmolll in EM and 265 I 30 mOsmoiil in ED). EM sperm that show low fertilizing capacity gave similarly poor results with insemination diluents at varying osmolalities (150 to 450 mOsmoV1). Furthemore, no relationship could be established between seminal plasma osmolality and low fertility for each individual. In tems of breeding management, seminal plasma osmolality cannot be retained as a critenon for selecting good EM males. Oocytes ageing in the abdominal cavity should be stnctly avoided for females reared in sea water.

Endogenous substrate for energy metabolism and ultrastructural correlates in spermatozoa of the sea urchin Diadema setosum.

Energy metabolism in spermatozoa of the sea urchin Diadema setosum of the order Diadematoida was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol, but also triglyceride (TG). Glycogen and glucose were present at extremely low levels. Following dilution of dry sperm and incubation in seawater, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for a slight increase in the level of free fatty acid. High lipase activity was demonstrated in the spermatozoa. 14C-labeled fatty acid was oxidized to 14CO2. Ultrastructural study also showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and some vacuoles appeared. Thus, the results obtained strongly suggest that D. setosum spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules at the midpieces.

Endogenous Substrates for Energy Metabolism in Spermatozoa of the Sea Urchins Arbacia lixula and Paracentrotus lividus.

Energy metabolism was examined in the spermatozoa of the sea urchins Arbacia lixula and Paracentrotus lividus, which belong to the orders Arbacioida and Echinoida respectively. P. lividus spermatozoa contained various phospholipids and cholesterol, and their endogenous triglyceride (TG) content was quite low. After dilution of dry sperm in artificial seawater, the level of phosphatidylcholine (PC) decreased rapidly, but other phospholipids remained at constant levels. In contrast to those of P. lividus, the spermatozoa of A. lixula contained TG as well as phospholipids and cholesterol. Following incubation of A. lixula spermatozoa in artificial seawater, TG decreased, but there were no concomitant changes in the levels of phospholipids. Trace amounts of glycogen were present in both species. High lipase activity was demonstrated in A. lixula spermatozoa, but in P. lividus spermatozoa lipase activity was low and phospholipase A2 activity was high. It is thus concluded that A. lixula spermatozoa obtain energy for swimming through oxidation of endogenous TG, whereas P. lividus spermatozoa use PC as a substrate for energy metabolism. This suggests that the system of energy metabolism in spermatozoa is different in the orders Arbacioida and Echinoida.

The Effects of Sperm Concentration, Sperm:Egg Ratio, and Gamete Age on Fertilization Success in Crown-of-Thorns Starfish (Acanthaster planci) in the Laboratory

Laboratory experiments varying gamete concentrations and gamete age demonstrated significant reductions in fertilization success of the starfish Acanthaster planci (L.) with decreasing sperm concentration and increasing age of both eggs and sperm. The effect of aging in sperm was faster than that of eggs, and the speed of sperm aging increased with increasing dilution of sperm. Fertilization success was high over a wide range of sperm: egg ratios but declined rapidly at ratios less than 50, particularly at low sperm concentrations. A. planci gametes aged more slowly, and the loss of fertilizing capacity of sperm with dilution (the respiratory dilution effect) was far less, than in sea urchins. These characteristics provide a mechanism for enhanced fertilization success at given sperm concentrations and at greater distances and times from the point of gamete release, and may explain the higher fertilization rates achieved over longer distances in the wild by A. planci relative to sea urchins. Gametes would remain competent for longer periods at more dilute concentrations and so better achieve long-distance fertilization. Gametes obtained at the end of the breeding season were qualitatively different from those obtained early in the breeding season and showed reduced fertilization success for a given combination of variables, and different fertilization dynamics.

Ionic factors affecting motility, respiration and fertilization rate of the sperm of the bivalvePecten maximus (L.)

Fertilization of the scallopPecten maximus occurs after gametes were naturally released in sea water by the bivalve which has undergone stimulation. The motility of the spermatozoa requires their dilution in sea water (1/40). Dilution triggers an immediate increase of oxygen consumption by sperm, reflecting an activation of a cyanide-sensitive respiration of a cellular origin. When scallops were stimulated by thermal shocks or by serotonin injection, sperm sampled at the urogenital pore output duct shows a respiration-motility activation after sea water dilution which is not seen in sperm scarified from the gonad. Dilution of kidney-sampled sperm into acidic (pH 5) or Na+-free artificial sea water reversibly inhibits both respiration and motility. In all cases fertilization rate of sperm is correlated to the increase of respiratory rate and motility measured after dilution in different media. Whether the scallop was stimulated or not, the pH of haemolymph and pericardic fluids were one pH unit below the value of sea water, the pH of the gonad and of the kidney tissues being more acidic (6.5 in average). Our results suggest that the acidic pH of the genital tract maintains the spermatozoa in a quiescent state and that capacitation occurs when male gametes move from the gonad to the kidney from where it is naturally released.

In vitro fertilization of pig oocytes matured in vitro

The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy.

Turbidity as a method of preparing sperm dilutions in the echinoid sperm/egg bioassay

The use of a turbidimeter for preparing sperm dilutions used in the echinoid sperm/egg bioassay was evaluated. Regression analyses of the relationship between sperm density and turbidity for the sea urchins Strongylocentrotus purpuratus and Strongylocentrotus droebachiensis and the sand dollar Dendraster excentncus indicated that although there were slope differences for each species, each coefficient of determination was highly significant (p ≤ 0.001). For Dendraster excentncus, triplicate hemacytometer counts over a range of turbidities as well as repeated preparations of a single sperm turbidity indicated similar variability for each. The use of the turbidimeter has time-saving advantages over conventional hemacytometer methods without sacrificing precision. Sperm dilutions can be prepared rapidly (1-2 min), minimizing sea water sperm preactivation before test initiation, and may therefore contribute to increased test precision.

Mating strategy and reproductive success of male patas monkeys (Erythrocebus patas)

Mating behavior and paternity of offspring of wild patas monkeys were studied at Kala Maloue National Park, Cameroon. Observation of patas groups over three years revealed that multi-male situations occurred after takeover of the position of a resident male. Direct observation of behavior showed that resident males (harem males) occupied only 31% of mating in multi-male situations and 100% in one-male situations. DNA-typing revealed that resident males sired two of four of infants in the one-male situation and four of five in the multi-male situation. Under the two years cycle of the one-male situation and the multi-male situation, calculation shows that resident males sired more offspring than sneakers both in observation and paternity testing. Sneak mating occurred during both one-male and multi-male situations, and resident males performed compensatory mating, with dilution of sneaker sperm; these activities explain the discrepancy found between observation of mating and results of paternity discrimination.