Giovanna Galeati

Resveratrol and Epigallocatechin-3-gallate addition to thawed boar sperm improves in vitro fertilization

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2xa0mM; Experiment 1) or EGCG (25, 50 or 100xa0μM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and inxa0vitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1xa0h of incubation at 37xa0°C. The addition of different doses of Res or EGCG to thawing extender for 1xa0h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (Pxa0<xa00.01) while only Res 2xa0mM induced a significant increase of this parameter (Pxa0<xa00.01). In addition, EGCG 25 and 50xa0μM supplementation significantly increased total fertilization efficiency as compared to control (EGCG 25xa0μM: 40.3xa0±xa08.2 vs 26.8xa0±xa09.5, Pxa0<xa00.05; EGCG 50xa0μM: 40.4xa0±xa07.8 vs 26.8xa0±xa09.5, Pxa0<xa00.01). The same effect was observed with Res 2xa0mM (51.0xa0±xa07.6 vs 29.6xa0±xa011.3, Pxa0<xa00.01). In conclusion, our results indicate that the addition of different doses of the two antioxidants to thawed spermatozoa for one hour, even if does not exert any effect on sperm viability and acrosome integrity, efficiently improves inxa0vitro penetration rate. Moreover, both molecules (EGCG 25 and 50xa0μM and Res 2xa0mM) significantly increases the total efficiency of fertilization.

Implementing an open-access CASA software for the assessment of stallion sperm motility: Relationship with other sperm quality parameters

Setting an open-access computer assisted sperm analysis (CASA) may benefit the evaluation of motility in mammalian sperm, especially when economic constraints do not allow the use of a commercial system. There have been successful attempts to develop such a device in Zebra fish sperm and the system has been used in very few studies on mammalian spermatozoa. Against this background, the present study aimed at developing an open-access CASA system for mammalian sperm using the horse as a model and based upon the Image J software previously established for Zebra fish sperm. Along with determining the sperm progressive motility and other kinetic parameters (such as amplitude of lateral head displacement), the results window was adjusted to simplify subsequent statistical analyses. The path window was enriched with colored sperm trajectories on the basis of the subpopulation they belong to and a number that allowed the sperm track to be associated to the sperm motility data shown in the results window. Data obtained from the novel plugin (named as CASA_bgm) were compared with those of the commercial CASA Hamilton-Thorn IVOS Vers.12, through Bland Altmans plots. While the percentage of total and progressive motile sperm, VCL, VAP, VSL, LIN and STR and ALH were in agreement with those obtained with the commercial system, BCF significantly differed between the two systems probably due to their settings. Interestingly, a positive and significant correlation between the percentages of total motile sperm evaluated through CASA_bgm and those showing high mitochondrial membrane potential evaluated by JC-1 staining was found. In conclusion, CASA_bgm ImageJ plugin could be useful and reliable for stallion sperm motility analysis and it is our aim to apply this system to other mammalian species.

Storage of sexed boar spermatozoa: Limits and perspectives.

Despite the great potential application of sex-sorted spermatozoa in swine, the technology is not practiced in the pig industry because of technical factors and species-specific issues. The susceptibility of boar spermatozoa to stresses induced by the sorting procedure, the relative slowness of the sex-sorting process together with the high sperm numbers required for routine artificial insemination in pig are some of the main factors limiting the commercial application of this technology in pigs. This review briefly describes the damage to spermatozoa during sex sorting, focusing on an additional limiting factor: increased susceptibility of sexed boar spermatozoa to injuries induced by liquid storage and cryopreservation that, in turn, impairs sperm quality leading to unsatisfactory results in vivo. Strategies to extend the lifespan of sex-sorted boar spermatozoa and to improve their fertilizing ability after liquid storage or cryopreservation need to be implemented before this technology can be used in pig farms. In this regard, encapsulation in barium alginate membranes could be a promising technique to optimize the in vivo use of sexed boar spermatozoa, by protecting, targeting, and controlling the release of sperm into the female genital tract.

Characterization of alkaline phosphatase activity in seminal plasma and in fresh and frozen-thawed stallion spermatozoa

Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen-thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is seminal plasma, whereas sperm extracts contribution is very low. In addition, we found that AP activity at physiological pH (7.0) is significantly lower than that observed at pH 8.0, including the optimal AP pH (pH 10.0). Alkaline phosphatase did not exert any effect on sperm-oocyte interaction assessed by heterologous oocyte binding assay. Additionally, we observed a thermal stability of seminal plasma AP, concluding that it is similar to that of bone isoforms. Positive correlations were found between seminal plasma AP activity and sperm concentration, whereas a negative correlation was present between both spermatozoa extracts and seminal plasma AP activity and seminal plasma protein content. A significant decrease in sperm extract AP activity was found in frozen-thawed samples compared with freshly ejaculated ones (nxa0=xa021), concomitantly with the decrease in sperm quality parameters. The positive correlation between seminal plasma AP activity measured at pH 10 and viability of frozen-thawed spermatozoa suggests that seminal plasma AP activity could be used as an additional predictive parameter for stallion sperm freezability. In conclusion, we provide some insights into AP activity in both seminal plasma and sperm extracts and describe a decrease in AP after freezing and thawing.

Pig sperm preincubation and gamete coincubation with glutamate enhance sperm-oocyte binding and in vitro fertilization

As the taste receptor for monosodium glutamate (umami) is expressed in both murine and human spermatozoa and the presence of α-gustducin and α-transducin, G proteins involved in the umami taste signaling, has been described in boar germ cells, the aim of this study was to evaluate if monosodium glutamate (MSG) would exert any effect on sperm-oocyte binding, inxa0vitro fertilization (IVF) and sperm parameters during inxa0vitro induced capacitation. For sperm-zona pellucida binding assay, boar spermatozoa were preincubated for 1xa0h and then coincubated for 1xa0h with denuded inxa0vitro matured oocytes in presence of different concentrations of MSG (0, 0.1, 1, 10xa0mM). MSG 1 and 10xa0mM significantly (Pxa0<xa00.05) increased the mean number of sperm bound to ZP compared with control (12.3xa0±xa09.0, 17.8xa0±xa011.3, 17.6xa0±xa010.8, MSG 0, 1 and 10xa0mM respectively). For inxa0vitro fertilization trials, both sperm preicubation (1xa0h) and gamete coincubation (1xa0h) were performed in presence of different concentrations of MSG (0, 0.1, 1, 10xa0mM). After 19xa0h of culture in fresh IVF medium, oocytes were fixed. MSG 1xa0mM significantly (Pxa0<xa00.05) increased the penetration rate compared with control (53.7xa0±xa020.4 vs. 36.8xa0±xa016.2). The addition of MSG during inxa0vitro induced capacitation of boar spermatozoa did not cause any significant difference, compared with control, on the percentage of viable cells, spermatozoa with intact acrosome and the percentage of spermatozoa displaying tyrosine-phosphorylation of sperm tail proteins. In order to evaluate whether the effect elicited by MSG could be due to glutamate uptake in boar spermatozoa, fertilization trials were performed in presence of either 1xa0mM MSG or 1xa0mM MSGxa0+xa0100xa0μM DL-threo-beta-hydroxyaspartic acid (THA), a non selective inhibitor of glutamate uptake. A significant increase (Pxa0<xa00.05) in the penetration rate in both MSG and MSGxa0+xa0THA groups compared to control was recorded (39.8xa0±xa015.7, 53.7xa0±xa022.1, 52.2xa0±xa023.7, Control, MSG and MSGxa0+xa0THA respectively) while no difference in penetration rate between MSG and MSGxa0+xa0THA treatment was observed suggesting that sperm glutamate transporters are not involved in the pathway mediating this effect. Our study demonstrates for the first time that glutamate exerts a positive effect on sperm-oocyte binding and fertilization. Further studies are needed to clarify the mechanism by which glutamate exert his effect.

Epigallocatechin-3-gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C.

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24-72xa0hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120xa0μm and green tea polyphenols, and p at .001, .01 and .1xa0mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48xa0hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48xa0hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48xa0hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24xa0hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.

Overstocking dairy cows during the dry period affects dehydroepiandrosterone and cortisol secretion

Stressful situations trigger several changes such as the secretion of cortisol and dehydroepiandrosterone (DHEA) from the adrenal cortex, in response to ACTH. The aim of this study was to verify whether overstocking during the dry period (from 21±3 d to the expected calving until calving) affects DHEA and cortisol secretion and behavior in Holstein Friesian cows. Twenty-eight cows were randomly divided into 2 groups (14 animals each), balanced for the number of lactations, body condition score, and expected date of calving. Cows in the far-off phase of the dry period (from 60 to 21 d before the expected calving date) were housed together in a bedded pack. Then, animals from 21±3 d before the expected calving until calving were housed in pens with the same size but under different crowding conditions due to the introduction of heifers (interference animals) into the pen. The control condition (CTR) had 2 animals per pen with 12.0m2 each, whereas the overstocked condition (OS) had 3 interference animals in the same pen with 4.8m2 for each animal. On d -30±3, -21±3, -15±3, -10±3, and -5±3 before and 10, 20, and 30 after calving, blood samples were collected from each cow for the determination of plasma DHEA and cortisol concentrations by RIA. Rumination time (min/d), activity (steps/h), lying time (min/d), and lying bouts (bouts/d) were individually recorded daily. In both groups, DHEA increased before calving and the concentration declined rapidly after parturition. Overstocking significantly increased DHEA concentration compared with the CTR group at d -10 (1.79±0.09 vs. 1.24±0.14 pmol/mL), whereas an increase of cortisol was observed at d -15 (3.64±0.52 vs. 1.64±0.46ng/mL). The OS group showed significantly higher activity (steps/h) compared with the CTR group. Daily lying bouts tended to be higher for the OS group compared with CTR group in the first week of treatment. The overall results of this study documented that overstocking during the dry period was associated with a short-term changes in DHEA and cortisol but these hormonal modifications did not influence cow behavior.

Biological effects of polyphenol-rich extract and fractions from an oenological oak-derived tannin on in vitro swine sperm capacitation and fertilizing ability

Although excessive ROS levels induce sperm damage, sperm capacitation is an oxidative event that requires low amounts of ROS. As the antioxidant activity of the ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, TanActiv R®) and its four fractions (FA, FB, FC, FD) has been recently reported, the present study was set up to investigate the biological effects of TRE and its fractions in an inxa0vitro model of sperm capacitation and fertilization. Boar sperm capacitation or gamete coincubation were performed in presence of TRE or its fractions (0, 1, 10, 100u202fμg/ml). TRE at the concentration of 10u202fμg/ml (TRE10) stimulated sperm capacitation, as it increased (pu202f<u202f.001) the percentage of spermatozoa with tyrosine-phosphorylated protein positivity in the tail principal piece (B pattern) (67.0u202f±u202f10.6 vs. 48.6u202f±u202f9.0, meanu202f±u202fSD for TRE10 vs. Ctr respectively). Moreover T10 significantly (pu202f<u202f.001) increased oocyte fertilization rate (91.9u202f±u202f4.0 vs. 69.0u202f±u202f14.8, TRE10 vs. Ctr respectively). An opposite effect of TRE at the concentration of 100u202fμg/ml (TRE100) on both sperm capacitation (B pattern cell percentage 33.3u202f±u202f29.2) and fertilizing ability (fertilization rate 4.9u202f±u202f8.3), associated with a higher sperm viability (66.9u202f±u202f9.3 vs. 35.4u202f±u202f10.8, TRE100 vs. Ctr respectively) (pu202f<u202f.001), was recorded. The potency of the TRE fractions seems to be highest in FB followed by FC, faint in FD and nearly absent in FA. Our results show that TRE and its fractions, in a different extent, exert a powerful biological effect in finely modulating capacitation and sperm fertilizing ability.

Alkaline phosphatase added to capacitating medium enhances horse sperm-zona pellucida binding

Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional role is not fully understood yet. Being that, sperm-oocyte interaction in equine species has been reported to be enhanced at a slightly basic pH, this work aimed at verifying whether exogenous alkaline phosphatase exerts any role on stallion spermatozoa and sperm-oocyte interaction at different pHs (7.4; 8.0; 9.0). Stallion spermatozoa were capacitated in Tyrodes medium at pH 7.4, 8.0, and 9.0 for 4xa0hours at 38xa0°C, 5% CO2 with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group); viability with mitochondrial activity, motility, and acrosome integrity were measured. In addition, a homologous binding assay was carried out: stallion spermatozoa were capacitated 1xa0hour at 38xa0°C, 5% CO2 with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group). Oocytes were then added to sperm suspensions and coincubated for 1xa0hour. Our results indicate that AP at pH 9.0 significantly increases the percentage of living cells with active mitochondria, whereas it significantly reduces the percentage of acrosome-damaged cells at pH 8.0. No significant differences were registered in motility parameters. The homologous binding assay showed a strong effect of AP, that increased the number of sperm bound to the oocytes zona pellucida at all pHs tested. In conclusion, AP can induce some modifications on sperm membranes thus enhancing their capacity to bind to the zona pellucida of equine oocytes.

Extended ex vivo culture of fresh and cryopreserved whole sheep ovaries

We describe an original perfusion system for the culture of whole ovine ovaries for up to 4 days. A total of 33 ovaries were divided into six groups: control (n=6), not perfused and fixed; Groups SM72 and SM72-FSH (n=6 each), perfused with a simple medium for 72h with or without FSH; Groups CM96 and CM96-FSH (n=6 each), perfused with a complex medium for 96h with or without FSH; Group CM96-FSH-cryo, (n=3) cryopreserved and perfused for 96h with Group CM96-FSH medium. Depending on the medium used, morphological parameters of cultured ovaries differed from fresh organs after 72 (SM72, SM72-FSH) or 96 (CM96, CM96-FSH) h of perfusion. Oestradiol and progesterone were secreted in all groups but FSH had an effect only on Group CM96-FSH, stimulating continued oestradiol secretion 10 times higher than in all other groups. Morphological parameters and hormone secretion of cryopreserved ovaries were not different from fresh controls. This method enables the culture of whole ovaries for up to 4 days, the time required in vivo for 0.5-mm follicles to grow to 2.2mm and then for these follicles to reach the ovulatory size of 4mm or more. It could be used as a research tool or to complement current techniques for preserving female fertility.