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Gamete co-incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co-incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production., (© 2014 Blackwell Verlag GmbH.)

The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen-thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen-thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC - ionomycin at 1.0μM, 0.1μM, 0.05μM or 0.01μM, or caffeine at 200μg/mL. The fertilising capacity of the semen was then assessed at 18h by staining the above oocytes with 4,6-diamidino-2-phenylindole (DAPI) and examining for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. In a second experiment, aliquots of frozen-thawed stallion sperm were subjected to DL-DGC selection - or not - and then further subjected to the two best capacitation treatments (0.1μM and 0.05μM ionomycin). The fertilising capacity of the semen was then determined as above. The DL-DGC/capacitated sperm samples showed the highest mean penetration rates: 24.16% following capacitation with 0.1μM ionomycin, and 12.21% following capacitation with 0.05μM ionomycin. The capacitated but non-DL-DGC-selected sperm returned significantly lower values: 6.26% and 7.02% for the same ionomycin treatments respectively. These findings suggest that combining DL-DGC selection with ionomycin capacitation improves the fertilising capacity of frozen-thawed stallion sperm., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The aims of the present study were: (1) to evaluate the influence of sperm concentration (ranging from 0.5 × 10(6) to 4 × 10(6) spermatozoa/ml) and length of the gamete co-incubation time (2, 4, 6, 8, 10, 12, 16, 20, 24 or 28 h) on in vitro fertilization (IVF), assessing the sperm penetration rate; (2) to investigate the kinetics of different semen parameters as motility, viability and acrosome status during the co-culture period; and (3) to analyse the effect of the presence of cumulus-oocytes complexes (COCs) on these parameters. To achieve these objectives, several experiments were carried out using in vitro matured oocytes from prepubertal goats. The main findings of this work are that: (1) in our conditions, the optimum sperm concentration is 4 × 10(6) sperm/ml, as this sperm:oocyte ratio (approximately 28,000) allowed us to obtain the highest penetration rate, without increasing polyspermy incidence; (2) the highest percentage of viable acrosome-reacted spermatozoa is observed between 8-12 h of gamete co-culture, while the penetration rate is maximum at 12 h of co-incubation; and (3) the presence of COCs seems to favour the acrosome reaction of free spermatozoa on IVF medium, but not significantly. In conclusion, we suggest that a gamete co-incubation for 12-14 h, with a concentration of 4 × 10(6) sperm/ml, would be sufficient to obtain the highest rate of penetration, reducing the exposure of oocytes to high levels of reactive oxygen species produced by spermatozoa, especially when a high sperm concentration is used to increase the caprine IVF outcome.

An experiment was designed to study the interaction between fresh/frozen-thawed donkey spermatozoa and zona pellucida (ZP)-free bovine oocytes in an attempt to develop a model for assessing cryopreserved Catalonian donkey sperm function. Semen from five donkeys was collected using an artificial vagina. Sperm motility and viability were immediately assessed and the semen sample cryopreserved. Sperm viability and motility were then reassessed immediately after thawing. The motion characteristics of the fresh and frozen-thawed spermatozoa were determined using a computer-assisted sperm analysis system. In vitro-matured cow oocytes were inseminated with different percent live donkey sperm (high (>60%) or low (<40%) viability donkey sperm). After 18h of co-incubation, the oocytes were fixed, stained with 4',6-diamidino-2-phenylindole (DAPI) and examined for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. Frozen-thawed spermatozoa from high viability semen showed significantly lower VCL, VAP and mean ALH values than did high viability fresh spermatozoa. In contrast, frozen-thawed spermatozoa of low viability had significantly higher velocity values than fresh spermatozoa of low viability. A significant positive correlation (P<0.01) was detected between percentage fertilization and viability (r=0.84), and between percentage fertilization and certain CASA parameters (VAP, r=0.56; VCL, r=0.61 and mean ALH, r=0.68). Fresh or frozen-thawed high viability spermatozoa penetrated 90.1% and 85.4% of bovine oocytes respectively. Lower rates of penetration were observed for fresh and frozen-thawed low viability spermatozoa (34% and 22.5% respectively). The donkey spermatozoa were able to fuse with the oolema and even to decondense and form the male pronucleus (85-94%). Larger numbers of penetrated spermatozoa per oocyte were recorded when high viability sperm samples were used, whether fresh (3.02 vs. 1.12 for low viability sperm) or frozen-thawed (3.41 vs. 1.47). Consequently, low viability sperm samples showed higher percentages of monospermic penetration (91.17% and 61.97% for fresh and frozen-thawed sperm samples respectively). These findings suggest that bovine oocytes provide a useful model for assessing the penetration potential of frozen-thawed donkey sperm., (Copyright 2009 Elsevier B.V. All rights reserved.)

In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [(14)C]glycogen after the addition to l-CCM with [(14)C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.

Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria green fluorescence protein/rat-liver glycogen synthase (GFP/LGS) resulted in the presence of GFP/GLS in spermatozoa from 7days to, at least, 16days after inoculation. The GFP/LGS was detected in the sperm heads after an "in vitro" fertilization procedure, either before or after the oocyte penetration. Our results indicate that spermatozoa carrying GFP/LGS protein conserved their fertilizing ability and were also detectable after oocyte penetration. This technique will allow to develop an easy system to follow the fate of mature sperm proteins.

Incubation of dog spermatozoa with 10 mmol l(-1) glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of L-lactate and CO2. In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l(-1)). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l(-1) to 10 mmol l(-1), fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.

The glycogen content in fresh raw dog spermatozoa was 0.22+/-0.03 micromol/mg protein. This matched with the presence of a glycogen-like staining in the head and midpiece. Glycogen levels lowered to 0.05 micromol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 micromol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35+/-0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25+/-0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo., (Copyright 2000 Wiley-Liss, Inc.)

The ability of adenoviral vectors to transfer DNA into boar spermatozoa and to offspring was tested. Exposure of spermatozoa to adenovirus bearing the E. coli lacZ gene resulted in the transfer of the gene to the head of the spermatozoa. Treatment did not affect either viability or acrosomal integrity of boar sperm. Of the 2-to 8-cell embryos obtained after in vitro fertilization with adenovirus-exposed sperm, 21.7% expressed the LacZ product. Four out of 56 piglets (about 7%) obtained after artificial insemination with adenovirus-exposed spermatozoa were positive in PCR analyses, even though none of the piglets showed the LacZ gene after southern blot analysis. RT-PCR analysis performed in tissues from two positive stillborn piglets showed the presence of the LacZ mRNA in all of the tissues tested. The offspring obtained after mating two positive animals did not show LacZ gene presence. Our results indicate that adenovirus could be a feasible mechanism for the delivery of DNA into spermatozoa, even though the transfer of the transgene may be limited to the first generation.

Experiments were carried out to develop an improved IVF system for prepubertal goat oocytes matured in vitro. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air. In Experiments 1 and 2, freshly ejaculated spermatozoa were capacitated in 1 of 3 media: TALP/H, modified Defined Medium (mDM) and mH-M199 with 50 micrograms/mL heparin for 45 min. Matured oocytes were fertilized in TALP, mDM or mH-M199 in Experiment 1 and in TALP in Experiment 2. In Experiment 3, three media were used for sperm capacitation and fertilization: Treatment A (control group): spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with 1 microgram/mL hypotaurine; Treatment B: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin + 388 micrograms/mL caffeine for 30 min and fertilized in TALP medium without hypotaurine; Treatment C: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with PHE (20 microM penicillamine, 10 microM hypotaurine and 2 microM epinephrine). At 24 h post insemination, the ova were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 8. In experiment 2, the results show, that mDM plus heparin for sperm capacitation and TALP medium with hypotaurine for oocyte fertilization provided the highest proportion of penetrated oocytes, both total number (79.6%) and normal fertilization (55.1%), whereas the use of caffeine (44.6 and 31.2%, total and normal fertilization rate, respectively) and PHE (31.8 and 20.6%, total and normal fertilization rate, respectively) as motility enhancers did not improve the results obtained in the control group (48.7% and 37.2%, total and normal fertilization rate, respectively). These were no differences for the results of morulae and blastocysts.

Contents Heat-shock protein 70 ( HSP70) plays a crucial role as intracellular cytoprotectant and molecular chaperone. A phenomenon of heat stress ( HS) leads to production of these proteins that could be beneficial to cells during cryopreservation, which is also a stressful process for the cell. This study aimed to evaluate the protective effect of exposure of bovine oocytes to moderate HS during in vitro maturation ( IVM) prior vitrification. First, oocytes were subjected to HS (41.5°C for 1 hr) at 0, 4, 8, 12 or 16 of IVM. Oocytes in vitro matured for 20 hr served as control group. Presence of HSP70 was detected at 20 hr by immunofluorescence. HSP70 expression was significantly higher when oocytes were subjected to HS at 8 hr of IVM. Next, oocytes were distributed into four groups: Control: IVM oocytes; VIT: oocytes vitrified/warmed at 20 hr of IVM; HS: oocytes subjected to HS at 8 hr of IVM; HS- VIT: oocytes subjected to HS at 8 hr of IVM and vitrified/warmed at 20 hr of IVM. Oocytes were fertilized at 24 hr of IVM, and cleavage and blastocyst yield were assessed. No significant differences were observed among treatments when cleavage rate was evaluated. However, fresh control and HS oocytes resulted in a significantly higher ( p < .05) blastocyst rate when compared to VIT and HS- VIT groups, although no significant differences within fresh or vitrified groups were observed. In conclusion, HS did not have a negative impact on the oocyte competence but HS applied before vitrification, offered no benefits for embryo development. [ABSTRACT FROM AUTHOR]