Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes.

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

The effects of glucuronic acid and N-acetyl-D-glucosamine on in vitro fertilisation of porcine oocytes.

High incidences of polyspermic penetration continue to challenge researchers during porcine in vitro fertilisation (IVF). The aim of this study was to reduce the incidence of polyspermy by increasing the perivitelline space thickness with glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) supplementation during oocyte maturation. After maturation, zona pellucida and perivitelline space thicknesses, intracellular glutathione concentrations and fertilisation kinetics were measured, in addition to embryonic cleavage and blastocyst formation at 48h and 144h after IVF, respectively. There were no significant differences between the treatments for zona pellucida thickness, penetration rates, male pronuclear formation or cortical granule exocytosis. Glucuronic acid supplementation significantly increased (PPPP<0.05) of cleavage and blastocyst formation by 48 and 144h after IVF compared with all other groups. These results indicate that supplementing with 0.005mM glucuronic acid and 0.005mM GlcNAc during oocyte maturation decreases the incidence of polyspermic penetration by increasing perivitelline space thickness and improving embryo development in pigs.

The effects of N-acetyl-L-cysteine supplementation on in vitro porcine oocyte maturation and subsequent fertilisation and embryonic development.

The effects of supplementation with 1.5 mM n-acetyl-l-cysteine (NAC) during in vitro oocyte maturation were studied. Oocytes were supplemented with 1.5 mM NAC during maturation for 0 to 24 h, 24 to 48 h, or 0 to 48 h then subjected to IVF and embryo development. Oocytes were evaluated after maturation for intracellular glutathione concentration, superoxide dismutase and glutathione peroxidase activities and DNA fragmentation. Fertilisation and embryonic development success were also evaluated. There was no effect of treatment on intracellular glutathione concentrations, enzyme activities or fertilisation success rates. Supplementing NAC during maturation significantly decreased (P < 0.05) the percentage of oocytes with fragmented DNA compared with no NAC supplementation. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of oocytes with male pronuclei than for oocytes from the other treatment groups. There was no difference in the percentage of embryos cleaved by 48 h after IVF between treatment groups. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of embryos reaching the blastocyst stage by 144 h after IVF compared with the other treatment groups. These results indicate that supplementation of the oocyte maturation medium with 1.5 mM NAC, specifically during the last 24 h, improves male pronucleus formation and blastocyst development in pigs.

Effects of dietary supplementation with an organic source of selenium on characteristics of semen quality and in vitro fertility in boars.

Semen characteristics in boars fed organic or inorganic sources of Se were assessed in 3 experiments. Crossbred boars were randomly assigned at weaning to 1 of 3 dietary treatments: I) basal diets with no supplemental Se (control), II) basal diets with 0.3 mg/kg of supplemental Se from an organic source (Sel-Plex, Alltech Inc., Nicholasville, KY), and III) basal diets supplemented with 0.3 mg/kg of supplemental Se from sodium selenite (Premium Selenium 270, North American Nutrition Co. Inc., Lewisburg, OH). For Exp. 1, semen was collected from boars (n = 10/dietary treatment) on 5 consecutive days at 15 mo of age. Effects of treatment × day were detected for the proportions of progressively motile (P = 0.02) and rapidly moving (P = 0.03) spermatozoa, and measures of sperm velocity, including path velocity of the smoothed cell path (P = 0.05) and average velocity measured in a straight line from the beginning to the end of the track (P = 0.05). Negative effects of day of semen collection on sperm motility were least pronounced in boars fed Sel-Plex. Experiment 2 was conducted when boars were 17 mo of age, and semen was collected (n = 10 boars/dietary treatment), diluted in commercially available extenders, and stored at 18°C for 9 d. Effects of treatment × day were detected for percentages of motile (P = 0.01) and static (P = 0.01) spermatozoa, amplitude of lateral head displacement (P = 0.02), frequency with which the sperm track crossed the sperm path (P = 0.04), straightness (P = 0.01), and average size of all sperm heads (P = 0.03). In general, sperm cells from boars fed Sel-Plex were better able to maintain motility during liquid storage compared with boars fed sodium selenite. For Exp. 3, semen was collected from boars (n = 6/dietary treatment) at 23 mo of age, and spermatozoa were evaluated at d 1 and 8 after semen collection using in vitro fertilization procedures. There was a tendency for an effect (P = 0.11) of dietary treatment on fertilization rate with Sel-Plex-fed boars having the greatest value (70.7%). The results of this study suggest that there are positive effects of dietary supplementation with Sel-Plex on boar semen characteristics and that organic Se supplementation may help ameliorate the negative effects of semen storage on characteristics of sperm motility.

N-acetyl-l-cysteine supplementation improves boar spermatozoa characteristics and subsequent fertilization and embryonic development.

The effects of 1.0 mmN-acetyl-l-cysteine (NAC) supplementation during the incubation of frozen-thawed and preserved boar sperm were studied in addition to subsequent oocyte IVF. Frozen-thawed and preserved boar sperm were supplemented with 1.0 mm NAC and incubated for 60 min to allow capacitation to occur followed by the addition of calcium ionophore 23187 to induce the acrosome reaction. The number of sperm having undergone the acrosome reaction was determined using the Wells-Awa staining technique. DNA damage was detected using single-cell gel electrophoresis. Membrane lipid peroxidation was estimated by the end point generation of malondialdehyde (MDA). Frozen-thawed sperm was not different in the ability of sperm to undergo the acrosome reaction but did have significantly (p < 0.05) more DNA damage (59.8 ± 1.0) compared to preserved sperm (32.0 ± 1.0%). Supplementing 1.0 mm NAC did not have an effect on the ability of sperm to undergo the acrosome reaction but did have significantly (p < 0.05) less DNA (39.2 ± 1.0%) damage compared to no antioxidant supplementation (52.7 ± 1.0%). Frozen-thawed sperm produced a significantly higher (p < 0.05) concentration of MDA (2.08 ± 0.05 µm MDA/10(7) cells) compared to preserved sperm (1.82 ± 0.05 µm MDA/10(7) cells), and non-supplemented sperm produced a significantly higher (p < 0.05) concentration of MDA (3.62 ± 0.05 µm MDA/10(7) cells) compared to the 1.0 mm NAC-supplemented sperm (0.28 ± 0.05 µm MDA/10(7) cells. Supplementation or semen storage method had no effect on IVF or embryonic development. These results indicate that supplementation with 1.0 mm NAC improved the ability to use frozen-thawed boar sperm during IVF as it reduces the DNA fragmentation and lipid peroxidation of the sperm.

Effects of N-acetyl-cysteine and N-acetyl-cysteine-amide supplementation on in vitro matured porcine oocytes.

This study was conducted to evaluate the effects of different concentrations of the antioxidant N-acetyl-cysteine (NAC) supplemented to the maturation medium on porcine embryo development. Concentrations of NAC and its synthetic derivative, NAC-amide (NACA) were evaluated for effects on nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mm) were supplemented to maturing oocytes, and embryo development was analysed at 48 and 144 h post-fertilization. There were no differences among cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mm NAC (56.5 ± 9.2%) was higher (p < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization or in cleavage rates when comparing 1.5 mm NAC and 1.5 mm NACA supplementation to the control. Blastocyst formation for 1.5 mm NAC (44.4 ± 4.7%) and 1.5 mm NACA (46.2 ± 3.4%) supplementation were higher (p < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mm of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development.

Mechanisms of oxidative stress in porcine oocytes and the role of anti-oxidants.

The mechanisms of oxidative stress in in vitro maturing porcine oocytes and the effects of anti-oxidant supplementation of the medium in ameliorating these effects were investigated in the present study. In addition to intracellular reduced glutathione (GSH) concentrations and DNA fragmentation, the present study focused on superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activity. The anti-oxidants used were N-acetylcysteine (NAC) and its derivative NAC-amide (NACA). The results indicate that when SOD is inhibited, supplementation of the maturarion medium with 1.5 mm NAC or NACA compensates for the decrease in SOD activity by reducing the degree of DNA fragmentation (P < 0.05). When GPx is inhibited, supplementation of the maturarion medium with 1.5 mm NAC alleviates the effects of no GPx activity, as indicated by a decrease in the degree of DNA fragmentation (P < 0.05). When the maturarion medium was supplemented with 1.5 mm NACA, intracellular GSH concentrations decreased (P < 0.05) and SOD and catalase activities increased (P < 0.05) along with the degree of DNA fragmentation. These results indicate that the mechanisms of alleviating oxidative stress in porcine oocytes are very complex and supplementing maturing oocytes with anti-oxidants may enhance enzyme activities and eliminate free radicals.

Characterization of a phospholipase Dalpha cDNA from tomato fruit.

Phospholipase D (PLD) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Total PL declines and phosphatidic acid increases in pericarp tissue during tomato fruit ripening, suggesting that increased PLD activity alters membrane structure. To assess the role of PLD in tomato ripening, we have begun a molecular genetic approach. Using a castor bean PLDalpha cDNA as a probe, a PLDalpha cDNA (LEPLD2) was isolated from our tomato fruit library. It has an open reading frame of 2421 nucleotides, encoding a polypeptide of 807 amino acids with a molecular mass of 92 kDa. The deduced LEPLD2 PLDalpha shares >75% sequence identity with PLDalphas from castor bean, tobacco and tomato. LEPLD2 transcript, detected by RNA gel-blot analysis, was very low in roots, low in stems, moderate in leaves, high in flowers, and increased in fruit during development and ripening. Expression of LEPLD2 in Escherichia coli yielded active enzyme, and a FLAG-PLDalpha fusion protein produced by transformed E. coli migrated close to the calculated 94 kDa on SDS/PAGE.

Steryl esters in the elaioplasts of the tapetum in developing Brassica anthers and their recovery on the pollen surface.

The tapetum cells in the developing anthers of Brassica napus contained abundant elaioplasts, which had few thylakoid membranes but were packed with globuli of neutral esters. Of the neutral esters, the major ester group possessed mainly 24-methylenecholesterol, 31-norcycloartenol, 24-dehydropollinastanol, and pollinastanol esterified to 18:3 and other unsaturated and saturated fatty-acyl moieties. The minor ester group had a dominant component tentatively identified as 12-dehydrolupeol esterified to mostly 18:0, 16:0, and 20:0 fatty-acyl moieties. The elaioplasts also contained a high proportion (16% w/w of total lipids) of monogalactosyldiacylglycerols (MGDG). This is the first report of plastids having steryl esters as the predominant lipids. We propose that the globuli contain steryl esters and are stabilized by surface MGDG and structural proteins. The tapetosomes, the other abundant lipid-containing organelles in the tapetum, possessed triacylglycerols (TAG) as the predominant lipids. At a late stage of anther development, the minor group of neutral esters and MGDG of the elaioplasts, as well as the TAG of the tapetosomes, were degraded. Steryl esters similar to those of the elaioplasts were recovered from the pollen surface and were the major lipids of the pollen coat. The pollen coat steryl esters and proteins could be extracted with moderately polar or nonpolar solvents. These proteins, which were mostly fragments of oleosins derived from the tapetosomes, had a high proportion of lysine (13 mol %). The possible functions of the steryl esters and the proteins on the pollen surface are discussed.