Lactoferrin increases sperm membrane functionality of frozen equine semen.

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 µg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 µM/µg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.

Milk, caseinate and lactoferrin addition to equine semen cooling extenders.

Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk- and glucose-based, C2) 0.6% caseinate, C3) C2 + Lf 200 µg ml-1 , C4) C2 + Lf 500 µg ml-1 and C5) C2 + Lf 1000 µg ml-1 extenders, and kept at 5 °C for 24 h. Sperm motility characteristics and intact membrane rates were not different among the treatments (P > 0.05). As a result of the cooling process, the nitrite concentration increased significantly in the cooled semen (69.6 ± 78.9 µm per ×106 spermatozoa) compared with the fresh semen (8.6 ± 1.9 µm per ×106 spermatozoa). In contrast, the H2 O2 concentrations were lower in the 0.6% caseinate extender (265.9 ± 221.3 µm per ×106 spermatozoa) than in the milk extender (430.9 ± 199.8 µm per ×106 spermatozoa, P < 0.05), showing an antioxidative effect of the caseinate compared with the milk. However, in all groups, hydrogen peroxide concentrations were similar to the undiluted fresh semen (332.8 ± 151.3 µm per ×106 spermatozoa). Caseinate showed to be as efficient as milk to protect equine-cooled spermatozoon.

Caseinate protects stallion sperm during semen cooling and freezing.

Extenders with a defined composition containing only components with clearly protective effects on sperm during storage would be an advantage. The aims of the present work were to assess whether caseinate, improves cooled and frozen equine semen quality. Semen from six stallions were suspended with four different cooling extenders C1) Kenney extender; C2) 0.6 % caseinate; C3) 2.7 % caseinate ; and C4) C1 + 2.1 % caseinate, and frozen extenders: F1) INRA 82 extender; F2) 1.35 % caseinate; and F3) 2.7 % caseinate. Although there was no significant difference between the motility rate among the cooled (C1:45.0, C2:36.7, C3:38.3 and C4:48.3) and frozen extenders (F1:16.9, F2:21.1 and F3:18.6), significant higher values of sperm velocity variables were observed with the 1.35 % caseinate extender compared to the control (VSL: 40.8 x 18.9 and VAP: 46.8 x 25.0 µm/s), respectively. Caseinate seemed to be responsible for sperm protection during preservation and showed to be as efficient as milk.

Addition of ficoll and disaccharides to vitrification solutions improve in vitro viability of vitrified equine embryo.

The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree C for 20s, the embryos were expelled out into 0.5M sucrose in PBS and transferred to PBS solution. The embryonic diameter was measured again and morphology and viability were evaluated with Propidium iodide and Hoechst 33258 dyes. Embryos vitrified with sucrose (19.2 percent) and trehalose (26.7 percent ) showed the highest percentage of viable cells and morphological quality.

Long-distance signaling within Coleus x hybridus leaves; mediated by changes in intra-leaf CO2?

Rapid long-distance signaling in plants can occur via several mechanisms, including symplastic electric coupling and pressure waves. We show here in variegated Coleus leaves a rapid propagation of electrical signals that appears to be caused by changes in intra-leaf CO2 concentrations. Green leaf cells, when illuminated, undergo a rapid depolarization of their membrane potential (Vm) and an increase in their apoplastic pH (pHa) by a process that requires photosynthesis. This is followed by a slower hyperpolarization of Vm and apoplastic acidification, which do not require photosynthesis. White (chlorophyll-lacking) leaf cells, when in isolated white leaf segments, show only the slow response, but when in mixed (i.e. green and white) segments, the rapid Vm depolarization and increase in pHa propagate over more than 10 mm from the green to the white cells. Similarly, these responses propagate 12-20 mm from illuminated to unilluminated green cells. The fact that the propagation of these responses is eliminated when the leaf air spaces are infiltrated with solution indicates that the signal moves in the apoplast rather than the symplast. A depolarization of the mesophyll cells is induced in the dark by a decrease in apoplastic CO2 but not by an increase in pHa. These results support the hypothesis that the propagating signal for the depolarization of the white mesophyll cells is a photosynthetically induced decrease in the CO2 level of the air spaces throughout the leaf.

Influência da infusäo transcervical de plasma seminal ou de estrógeno na concepçäo, no ciclo estral e na ovulaçäo de porcas / Influence of transcervical infusion of seminal plasma or oestrogen on conception, oestrus cycle and ovulation in sows

O objetivo deste trabalho foi o de avaliar o desempenho de 211 porcas da linhagem Camborough 22 Marca Registrada, de diferentes ordens de parto, submetidas à infusäo transcervical de plasma seminal ou de estrógeno no início do estro. Após o desmame, elas foram examinadas duas vezes ao dia para a detecçäo do estro, na presença de um macho sexualmente maduro, e da ovulaçäo, com auxílio da ultrasonografia transcutânea. No início do estro as fêmeas receberam aleatoriamente uma infusäo transcervical de plasma seminal, de estrógeno ou permaneceram como grupo-controle. Todas as fêmeas foram inseminadas no turno seguinte à detecçäo do estro, recebendo no máximo três inseminaçöes com intervalos de 8 a 16h. No grupo-controle as fêmeas de primeiro parto ovularam mais precocemente que as fêmeas com maior número de partos (P<0,05). Os tratamentos näo influenciaram a duraçäo do estro e o momento da ovulaçäo. O número total de leitöes nascidos näo diferiu entre os tratamentos

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars.

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n=14 gilts) and heterospermic (n=11 gilts) insemination. Single AI's were performed under suboptimal conditions using 1 x 10(9) spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P<0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P<0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.

Chlorophyll is not the primary photoreceptor for the stimulation of P-type H+ pump and growth in variegated leaves of Coleus x hybridus.

There has been persisting controversy over the role of photosynthesis in the stimulation of the plasma membrane H+-ATPase and growth of dicotyledonous leaves by light. To investigate this, we compared the effects of light on growth, H+ net efflux and membrane potential (Vm) of strips which contained either only chlorophyll-free (white) mesophyll cells or chlorophyll-containing (green) cells cut from variegated Coleus leaves. White mesophyll cells responded to white, blue and red light with a hyperpolarization of Vm, an acidification of the apoplast and a promotion of growth, all of which began after a lag of 2-7 min. In contrast, green mesophyll cells showed a biphasic light response in which the hyperpolarization and the acidification were preceded by a rapid depolarization of Vm and an alkalinization of the apoplast. Nevertheless, green and white tissues showed comparable growth promotions in response to light. The light response of the leaf mesophyll is a composite of two separate photosystems. The initial depolarization and alkalinization are mediated by photosynthesis and blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The slower hyperpolarization, acidification and growth response, on the other hand, are clearly in response to light absorption by pigments other than chlorophyll.

The Propagation of Slow Wave Potentials in Pea Epicotyls.

Slow wave potentials are considered to be electric long-distance signals specific for plants, although there are conflicting ideas about a chemical, electrical, or hydraulic mode of propagation. These ideas were tested by comparing the propagation of hydraulic and electric signals in epicotyls of pea (Pisum sativum L). A hydraulic signal in the form of a defined step increase in xylem pressure (Px) was applied to the root of intact seedlings and propagated nearly instantly through the epicotyl axis while its amplitude decreased with distance from the pressure chamber. This decremental propagation was caused by a leaky xylem and created an axial Px gradient in the epicotyl. Simultaneously along the epicotyl surface, depolarizations appeared with lag times that increased acropetally with distance from the pressure chamber from 5 s to 3 min. When measured at a constant distance, the lag times increased as the size of the applied pressure steps decreased. We conclude that the Px gradient in the epicotyl caused local depolarizations with acropetally increasing lag times, which have the appearance of an electric signal propagating with a rate of 20 to 30 mm min-1. This static description of the slow wave potentials challenges its traditional classification as a propagating electric signal.

Mannitol inhibits growth of intact cucumber but not pea seedlings by mechanically collapsing the root pressure.

The positive xylem pressure (Px) in cucumber hypocotyls is a direct extension of root pressure and therefore depends on the root environment. Solutions of the electrolyte KCl (0-10 osm) reduced the hypocotyl Px transiently (biphasic response), while the Px reduction by mannitol solutions was sustained. The amplitudes of the induced Px reduction depended directly, and the degree of Px restoration after stress release depended indirectly, on the size of the initial positive Px indicating that mannitol released the root pressure by a mechanical rather than osmotic mechanism. Mannitol treatment and other means of root pressure reduction revealed two separate growth responses in the affected cucumber hypocotyls. Only steep Px drops (following root excision or root pressure release in mannitol) directly cause a rapid, transient drop in growth rate (GR). Both rapid and slow (after root incubation in KCN or NEM) decreases in root pressure, however, led to a sustained growth inhibition of cucumber hypocotyls after about 30 min. This delay characterizes the growth response as an indirect consequence of the Px change. Pea seedlings, which lacked root pressure and had a negative Px throughout, showed extremely small changes in epicotyl Px and GR after root incubation in mannitol. It is apparent that the higher sensitivity of cucumber growth to mannitol depended on the presence and release of root pressure.

Induction and ionic basis of slow wave potentials in seedlings of Pisum sativum L.

Slow wave potentials (SWPs) are transient depolarizations which propagate substantial distances from their point of origin. They were induced in the epidermal cells of pea epicotyls by injurious methods such as root excision and heat treatment, as well as by externally applied defined steps in xylem pressure (Px) in the absence of wounding. The common principle of induction was a rapid increase in Px. Such a stimulus appeared under natural conditions after (i) bending of the epicotyl, (ii) wounding of the epidermis, (iii) rewatering of dehydrated roots, and (iv) embolism. The induced depolarization was not associated with a change in cell input resistance. This result and the ineffectiveness of ion channel blockers point to H(+)-pumps rather than ion channels as the ionic basis of the SWP. Stimuli such as excision, heat treatment and pressure steps, which generate SWPs, caused a transient increase in the fluorescence intensity of epicotyls loaded with the pH-indicator DM-NERF, a 2',7'-dimethyl derivative of rhodol, but not of those loaded with the pH indicator 2',7'bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Matching kinetics of depolarization and pH response identify a transient inactivation of proton pumps in the plasma membrane as the causal mechanism of the SWP. Feeding pump inhibitors to the cut surface of excised epicotyls failed to chemically simulate a SWP; cyanide, azide and 2,4-dinitrophenol caused sustained, local depolarizations which did not propagate. Of all tested substances, only sodium cholate caused a transient and propagating depolarization whose arrival in the growing region of the epicotyl coincided with a transient growth rate reduction.

Surface plasmon resonance detection and multispot sensing for direct monitoring of interactions involving low-molecular-weight analytes and for determination of low affinities.

Surface plasmon resonance detection allows direct observation of molecular interactions between an analyte in solution and its immobilized binding partner. The use of simultaneous monitoring of interaction events on multiple sensing surfaces, with varying amounts of immobilized receptor, for detection of low-molecular-weight analytes and for determination of low affinities was investigated. Using multispot sensing and BIAcore 2000 instrumentation, analytes as small as 180 Da were detected and affinities in the 50 microM range could be determined. The use of multispot sensing thus extends direct interaction analysis to include low-molecular-weight analytes and low-affinity interactions.

Comparison of electric and growth responses to excision in cucumber and pea seedlings. II. Long-distance effects are caused by the release of xylem pressure.

Excision of a growing stem causes local wound responses, such as membrane depolarization and growth inhibition, as well as effects at larger distances from the cut. In this study, cucumber hypocotyls were excised 100 mm below the hook, so that the growing region was beyond the reach of the wound-induced depolarization (up to 40 mm). Even at such a distance, the cut still caused a considerable and rapid drop in the hypocotyl growth rate. This growth response is not a direct wound response because it does not result from the cut-induced depolarization and because it can be simulated by root pressure manipulation (using a pressure chamber). The results indicate that the growth response resulted from the rapid release of the xylem pressure upon excision. To test this conclusion we measured the xylem pressure by connecting a pressure probe to the cut surface of the stem. Xylem pressure (Px) was found to be +10 to +40 kPa in cucumber hypocotyls and -5 to -10 kPa or lower in pea epicotyls. Excision of the cucumber hypocotyl base led to a rapid drop in Px to negative values, whereas excision in pea led to a rapid rise in Px to ambient (zero) pressure. These fast and opposite Px changes parallel the excision-induced changes in growth rate (GR): a decrease in cucumber and a rise in pea. The sign of the endogenous xylem pressure also determined whether excision induced a propagating depolarization in the form of a slow wave potential (SWP). Under normal circumstances pea seedlings generated an SWP upon excision whereas cucumber seedlings failed to do so. When the Px in cucumber hypocotyls was experimentally inverted to negative values by incubating the cumber roots in solutions of NaCN or n-ethylmaleimide, excision caused a propagating depolarization (SWP). The experiment shows that only hydraulic signals in the form of positive Px steps are converted into propagating electric SWP signals. These propagating depolarizations might be causally linked to systemic 'wound' responses, which occur independently of the short-distance or direct wound responses.

Comparison of electric and growth responses to excision in cucumber and pea seedlings. I. Short-distance effects are a result of wounding.

The local electric response to stem excision in both pea epicotyls and cucumber hypocotyls is a depolarization of the cells in the wound area. If we define wound area as the region of local depolarization, we find that it extends for approximately 10 mm from the cut or wound site in pea epicotyls, whereas it can reach up to 40 mm in cucumber hypocotyls. The wound-induced depolarization in pea cells is transient, reaching its maximal amplitude within 1-2 min, whereas in cucumber cells this depolarization is more sustained. A third difference between wound responses in pea and cucumber is the intermittent appearance of spikes, i.e. very short, rapidly reverted depolarizations which frequently accompany the basic depolarization in cucumber but not in pea cells. These spikes can propagate in both directions along the hypocotyl axis. The cause of the different responses of pea and cucumber cells is unknown. A possible explanation might be found in different degrees of electrical cell coupling in the two species. This possibility was investigated in cucumber hypocotyls by measuring the cell input resistance (R(in)) of epidermal cells at various axial distances from the cut. Shorter distances increase the likelihood of shunting the cell membrane resistance through the shortened symplastic path to the cut surface. With a series of cuts made at decreasing distances from the measured site, cell depolarization increased without comparable changes in R(in). Two conclusions were drawn. Firstly, wound-induced depolarizations are not brought about by shunting of the cell resistance in the wound area. Secondly, the depolarization is probably not carried by ion channels but may be caused by an inhibition of proton pump activity. Parallel to its depolarization effect on the membrane potential, excision led to a severe and sustained decline in the cucumber hypocotyl growth rate only when carried out sufficiently close to the growing region (45 mm from the hook). Similar excision in pea epicotyls failed to change the growth rate. Both electrical and growth data support the concept that the high and sustained responsiveness of cucumber seedlings to wounding is caused by a particular sensitivity of their proton pump mechanism.

Rapid alterations in growth rate and electrical potentials upon stem excision in pea seedlings.

Excision of the epicotyl base of pea (Pisum sativum L.) seedlings in air results in a fast drop in the growth rate and rapid transient membrane depolarization of the surface cells near the cut. Subsequent immersion of the cut end into solution leads to a rapid, transient rise in the epicotyl growth rate and an acropetally propagating depolarization with an amplitude of about 35 mV and a speed of approx. 1 mm s-1. The same result can be achieved directly by excision of the pea epicotyl under water. Shape, amplitude and velocity of the depolarization characterize it as a "slow-wave potential". These results indicate that the propagating depolarization is caused by a surge in water uptake. Neither a second surge in water uptake (measured as a rapid increase in growth rate when the cut end was placed in air and then back into solution) nor another cut can produce the depolarization a second time. Cyanide suppresses the electrical signal at the treated position without inhibiting its transmission through this area and its development in untreated parts of the epicotyl. The large depolarization and repolarization which occur in the epidermal and subepidermal cells are not associated with changes in cell input resistance. Both results indicate that it is a transient shut-down of the plasma-membrane proton pump rather than large ion fluxes which is causing the depolarization. We conclude that the slow wave potential is spread in the stem via a hydraulic surge occurring upon relief of the negative xylem pressure after the hydraulic resistance of the root has been removed by excision.