Acrosome Integrity Research Articles

Minimizing sperm oxidative stress using nanotechnology for breeding programs in rams

BackgroundArtificial insemination (AI) is a routine breeding technology in animal reproduction. Nevertheless, the temperature-sensitive nature and short fertile lifespan of ram sperm samples hamper its use in AI. In this sense, nanotechnology is an interesting tool to improve sperm protection due to the development of nanomaterials for AI, which could be used as delivery vehicles. In this work, we explored the feasibility of vitamin E nanoemulsion (NE) for improving sperm quality during transport.ResultsWith the aim of evaluating this proposal, ejaculates of 7 mature rams of Manchega breed were collected by artificial vagina and extended to 60 × 106 spz/mL in Andromed®. Samples containing control and NE (12 mmol/L) with and without exogenous oxidative stress (100 µmol/L Fe2+/ascorbate) were stored at 22 and 15 ºC and motility (CASA), viability (YO-PRO/PI), acrosomal integrity (PNA-FITC/PI), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) monitored during 96 h. Our results show that NE could be used to maintain ram spermatozoa during transport at 15 and 22 ºC for up to 96 h, with no appreciable loss of kinematic and physiological characteristics of freshly collected samples.ConclusionsThe storage of ram spermatozoa in liquid form for 2–5 d with vitamin E nanoemulsions may lead more flexibility to breeders in AI programs. In view of the potential and high versatility of these nanodevices, further studies are being carried out to assess the proposed sperm preservation medium on fertility after artificial insemination.Graphical

Bovine sperm HSP-70 molecules: a potential cryo-tolerance marker associated with semen quality and fertility rate.

Freezability is the ability of sperm to maintain its vitality and quality from various stress during the cryopreservation process, which is very important for the success of fertilization in AI programs. Heat shock proteins (HSPs) are unique proteins induced in response to various stress, including excess reactive oxygen species (ROS) and oxidative damage to intracellular enzymes that can harm cells. This study aimed to analyze the potential of HSP-70 molecules in bovine sperm as a marker of freezability or cryo-tolerance, as well as its association with semen quality and fertility rate. The classification of bulls is based on freezability (good freezability/GF and poor freezability/PF), which is obtained from the value of post-thaw viability using the SYBR-14/PI-flow cytometry. Semen quality assessed included sperm motility and kinetics (computer-assisted sperm analyses), plasma membrane integrity (HOS test), acrosome integrity (FITC-PNA), mitochondrial membrane (JC-1), and DNA damage (Halomax kit). The bull fertility rate assessment was analyzed based on the first service conception rate of each bull derived from data on the success of artificial insemination contained in the Indonesian-integrated National Animal Health Information System (iSIKHNAS). Gene expression levels of HSP-70 bovine sperm were performed using the RT-qPCR method. The protein abundance of HSP-70 bovine sperm was determined using the enzyme immunoassay (EIA) method. Bovine sperm HSP-70 molecules, at the gene and protein level, showed a higher abundance in GF (p < 0.05) than in PF bulls. The percentage of each parameter of frozen-thawed sperm quality was significantly higher in GF (p < 0.05) than in PF bulls. The HSP-70 molecules at the gene and protein levels were significantly positively correlated (p < 0.01) with the fertility rate. Furthermore, HSP-70 molecules were negatively associated (p < 0.01) with low mitochondrial membrane potential and sperm DNA damage and positively correlated (p < 0.01) with other frozen-thawed sperm quality parameters. The overall quality of frozen-thawed sperm was closely related (p < 0.01) to the fertility rate. We may conclude that HSP-70 molecules in bovine sperm at the gene and protein level have the potential to be developed as a marker for cryo-tolerance or freezability, which may be utilized as a predictor of fertility and frozen-thawed sperm quality in bulls.

Effect of season on the dynamics of cat sperm DNA fragmentation

BackgroundFeline species undergo reproductive seasonality; thus, sperm characteristics, such as DNA integrity, can be affected by the photoperiod. This study was conducted to determine the effect of seasonal changes on sperm quality and on the dynamics of sperm DNA fragmentation. Epididymal spermatozoa were collected from 36 tomcats subjected to bilateral orchiectomy during breeding (BS) and non-breeding (NBS) seasons. Sperm samples were obtained by cutting the cauda epididymis and assessed for sperm motility, concentration, acrosome integrity, plasma membrane integrity and sperm morphology. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test after 0, 6, and 24 h of incubation at 37 °C.ResultsThe total sperm motility and plasma membrane integrity values were greater during the BS, while the percentages of abnormal sperm and head defects were lesser (p < 0.05). No significant differences in DNA fragmentation were found between seasons after sperm collection. DNA damage was greater after 24 h of incubation at 37 °C in both seasons, although the percentage of spermatozoa with fragmented DNA was significantly lesser in the BS than in the NBS at 24 h (p < 0.05).ConclusionsThe study suggests seasonal changes in some of the quality parameters of cat sperm. DNA fragmentation dynamics were affected by the time of incubation and reproductive season; therefore, this technique might be used as an additional tool to test the potential fertility of semen samples used in feline-assisted reproduction.

Mito-Tempo improves acrosome integrity of frozen-thawed epididymal spermatozoa in tomcats.

In tomcats, epididymal spermatozoa provide an additional source of male gametes available for cryopreservation. While this procedure is feasible, the survival rate and motility of epididymal cat spermatozoa are both low after thawing. Cryopreservation is known to induce oxidative stress in spermatozoa, with mitochondria and the plasma membrane being the two major generation sites, and an imbalanced presence of free radicals is a possible cause for this low survival rate. Different antioxidants have been tested before for their effect on cryopreserved cat spermatozoa quality, with varying results. Here, we used Mito-Tempo, which is a synthetic mitochondria-targeted antioxidant and a specific scavenger of the mitochondrial superoxide system. By supplementing Mito-Tempo with the freezing extender, we aimed to improve the sperm quality of frozen-thawed cat epididymal spermatozoa. Epididymal spermatozoa obtained from twelve tomcats were assessed for motility and concentration. Prior to freezing, samples were diluted in TRIS buffered extender with egg yolk and glycerol and divided into five aliquots supplemented with 0 (control), 0.5, 5, 50, and 1005M of Mito-Tempo. After thawing, sperm motility, concentration, morphology, plasma membrane integrity, acrosome integrity, and mitochondrial membrane potential were evaluated. A Friedman rank sum test with a Bonferroni post-hoc test was used to determine statistical in-between group differences in post-thaw semen parameters. The results indicated a slight improvement in acrosome integrity across all groups that were supplemented with Mito-Tempo, with the group that received 55M of Mito-Tempo showing the greatest improvement [(median of 67.99%, IQR of 5.55) compared to the control group (median of 65.33%, IQR of 7.75; P = 0.05)]. For all other sperm parameters, no significant differences (P > 0.05) were detected between different Mito-Tempo concentrations. These findings highlight the protective effect of Mito-Tempo on acrosome integrity and suggest that 55M is the most effective concentration for maintaining acrosome integrity. Since Mito-Tempo has shown a positive effect on multiple sperm parameters in other species, such as men, boars, roosters, rams, and bulls, we need to conclude that species-specificity may play a role here.

Cryopreservation and freezability of epididymal and ejaculated stallion spermatozoa

The aim of the present study was to compare the seminal attributes and freezability of the epididymal sperm recovered from castrated stallions and ejaculated semen collected through artificial vagina from the same stallions previously. The seminal quality parameters viz., viability, sperm morphology, plasma membrane functional integrity, acrosome integrity and mitochondrial membrane potential were studied, and recorded immediately after fresh semen collection/retrieval, before freezing, and after cryopreservation. Similar protocols were followed for freezing the epididymal and ejaculated semen. From the current study, it was observed that the sperm concentration of the epididymal recovered was significantly higher than that of fresh ejaculation. Other seminal parameters in thefresh sample, like total and progressive sperm motility, sperm membrane functional integrity, acrosome integrity and kinematic parameters like VSL, STR, and linearity were similar without any significant differences between both. However, mitochondrial membrane potential, BCF and linearity of the spermatozoa differed significantly between the groups after cryopreservation. From the current study, it can be concluded that the protocols used for cryopreserving the fresh ejaculates can be adopted for freezing the stallion epididymal spermatozoa without causing significant damage and through this method; elite stallion germplasm can be safely cryopreserved and conserved.

Cryopreservation and freezability of epididymal and ejaculated stallion spermatozoa

The aim of the present study was to compare the seminal attributes and freezability of the epididymal sperm recovered from castrated stallions and ejaculated semen collected through artificial vagina from the same stallions previously. The seminal quality parameters viz., viability, sperm morphology, plasma membrane functional integrity, acrosome integrity and mitochondrial membrane potential were studied, and recorded immediately after fresh semen collection/retrieval, before freezing, and after cryopreservation. Similar protocols were followed for freezing the epididymal and ejaculated semen. From the current study, it was observed that the sperm concentration of the epididymal recovered was significantly higher than that of fresh ejaculation. Other seminal parameters in thefresh sample, like total and progressive sperm motility, sperm membrane functional integrity, acrosome integrity and kinematic parameters like VSL, STR, and linearity were similar without any significant differences between both. However, mitochondrial membrane potential, BCF and linearity of the spermatozoa differed significantly between the groups after cryopreservation. From the current study, it can be concluded that the protocols used for cryopreserving the fresh ejaculates can be adopted for freezing the stallion epididymal spermatozoa without causing significant damage and through this method; elite stallion germplasm can be safely cryopreserved and conserved.

Exogenous progesterone during in vitro fertilization improves developmental competence of partially cumulus-denuded bovine oocytes

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus-denuded oocytes in serum-free conditions. After a 4-h capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 μM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases. Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis, which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 μM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22 h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 μM P4 led to inefficient IVF outcomes (P < 0.05). Therefore, IVF trials with partially cumulus-denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 μM). Cleavage rate significantly increased at 1 μM P4, which translated to increased total embryo production after 7 days of in vitro culture (P < 0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 μM and 10 μM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 μM during IVF improved the developmental competence of partially cumulus-denuded oocytes in serum-free conditions.

Effect of Ferulic Acid on Semen Quality of Goat Bucks during Liquid Storage at 17 °C.

This study investigated the effect of different concentrations of ferulic acid (FA) on the quality of goat semen preserved at 17 °C. First, semen was collected from three black-headed goat bucks using an artificial vagina. Then, the mixed semen was diluted with basal dilutions containing different concentrations of FA (0, 25, 50, 100, and 200 μmol/L) and stored at 17 °C. Sperm total motility, plasma membrane integrity, acrosome integrity, reactive oxygen species (ROS) levels, malondialdehyde (MDA) content, and total antioxidant capacity (T-AOC) were measured during semen storage. The results showed that sperm total motility, plasma membrane integrity, and acrosome integrity were significantly improved in the 50 μmol/L FA group compared with the control group (0 μmol/L) on days 1-5, and the level of T-AOC significantly increased, while the contents of ROS and MDA significantly reduced. Meanwhile, the goats' conception rate showed that supplementing semen with 50 μmol/L FA preserved at 17 °C for 3 days had no significant effect on fertility. Taken together, our findings suggest that adding 50 μmol/L FA in dilution at 17 °C can improve goat bucks' semen quality.

Myo-inositol improves the viability of boar sperm during liquid storage.

Liquid preservation of boar semen is a highly preferred method for semen preservation in pig production. However, oxidative stress is the main challenge during the liquid preservation of boar semen in a time dependent manner. Therefore, supplementation of sperm with antioxidants during storage to protect them from oxidative stress has been the focus of recent research. Myo-inositol (Myo-Ins), the most active form of inositol, which belongs to the vitamin (Vit.) (B1 group has been shown to improve semen quality) (1). This study aimed to investigate whether Myo-Ins supplementation protects boar sperm in liquid preservation against oxidative stress and determine the appropriate concentration of Myo-Ins to be used in this regard. Boar sperm was diluted with a semen extender with different concentrations of Myo-Ins (2, 4, 6, and 8 mg/mL) depending on the previous studies (1, 24). Sperm motility and viability, plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), semen time survival, and gene expression were measured and analyzed on days 0, 1, 3, 5, and 7 for the different samples. Different concentrations of Myo-Ins exerted different protective effects on the boar sperm quality. The addition of 2 mg/mL Myo-Ins resulted in higher sperm motility and viability, plasma membrane and acrosome integrity, MMP, and effective survival time. Investigation of mRNA expression patterns via qRT-PCR suggested that the 2 mg/mL Myo-Ins sample had increased expression of antioxidative genes. The addition of Myo-Ins to semen extender improved the boar semen quality by decreasing the effects of oxidative stress during liquid preservation at 17°C. Additionally, 2 mg/mL is the optimum inclusion concentration of Myo-Ins for semen preservation.

Semen characteristics, nutrient utilization and immuno-endocrine status in response to dietary supplementation of a novel trace mineral mixture in crossbred bulls

BackgroundMicronutrients have a well-established role in the productive and reproductive health of animals, and there are many proven benefits of supplementing them in the diet of bulls or semen extender per se. Trace minerals have a key role in regulating male fertility and sperm function. MethodologyTwelve crossbred bulls were divided into two groups (n = 6): T1(control) and T2, (BullTraMin) and given diets with or without supranutritional supplementation of the novel trace mineral (BullTraMin), respectively. The experiment was conducted for 120 days and the effects on nutrient utilization, immuno-endocrine status, sexual behaviour and semen characteristics were investigated. Results and conclusionResults showed that body weight, dry matter intake, and nutrient digestibility were not affected by dietary supplementation with BullTraMin. Testosterone levels were increased (p < 0.05), while thyroxine hormone concentrations were unaffected (p < 0.05) in control (T1) and supplemented (T2) groups. Levels of trace minerals (Zn, Cu, and I) were improved in the blood and seminal plasma (p < 0.05) of the T2 group, while levels of Cr and Mn were increased only in the blood plasma. Sperm functions viz., viability, membrane integrity, acrosome integrity, motility, and mitochondrial membrane potential were improved (p < 0.05) due to BullTraMin supplementation. Furthermore, lipid peroxidation and membrane scrambling which impair the cryopreservilibity of spermatozoa, were reduced (p < 0.05) in T2 group. Blood antioxidant status (total antioxidant activity, superoxide dismutase and glutathione peroxidase levels) was improved (p < 0.05) in T2 group. Sexual behaviour was also improved (p < 0.05) in BullTraMin-supplemented group. Hence, it can be concluded that BullTraMin supplementation at a dose rate of 889 mg/kg diet (dry matter intake) is beneficial in improving hormonal status, antioxidant status, sexual behaviour and semen quality.

Soybean lecithin and cholesterol-loaded cyclodextrin in combination to enhances the cryosurvival of dairy goat semen

The objective of the study was to examine the effect of soy lecithin (SL) and cholesterol loaded cryclodestrin (CLC) on cryo-survival of sperm cryopreserved in the presence or absence of seminal plasma in Saanen dairy goats. Tris-based dilutions containing various concentrations of SL (0, 0.5%, 1.0% or 2.0%) and CLC (0, 2.0 g/L, 4.0 g/L or 6.0 g/L CLC) were used to cryopreserve Saanen dairy goat sperm. The quality of frozen-thawed sperm, including progressive motility, viability, acrosome and plasma membrane integrity, as well as fertility were detected. Results found that the optimal combination of the two cryoprotectants was 1.0% SL+4.0 g/L CLC, which significantly increased progressive motility, viability, acrosome and plasma membrane integrity of frozen thawed sperm. The impact of the two cryoprotectants in combination was not affected by the presence of seminal plasma. The conception rates obtained after artificial insemination using sperm cryopreserved with and without seminal plasma were 88.89% and 91.67% (P > 0.05), respectively. The respective values for average number of litter sizes were 1.55 ± 0.17 and 1.56 ± 0.21 (P > 0.05). Therefore, this study improved the cryopreservation efficiency of goat semen, enhanced the sperm cryosurvival, and layed a foundation for the wide application of frozen goat semen.

Influence of Equilibration Time and Bull-Specific Extender for Cryopreservation on Semen Quality and Fertility in German Holstein Friesian Bulls: A Controlled Field Trial.

Equilibration with an extender is necessary to allow cryopreservation of bovine sperm. The aim of trial 1 was to assess the effect of 24 h versus 4 h equilibration time with three different extenders on sperm quality and to select the preferred extender for each bull. The aim of trial 2 was to investigate the effect of using a 24 h equilibration time with a bull-specific extender on field fertility. For trial 1, three ejaculates each from eight Holstein Friesian breeding bulls were used as the split-sample, including two equilibration times (4 h and 24 h) and three extenders (BioXcell, Triladyl, and OptiXcell). For trial 2, from 5 to 10 ejaculates from the same bulls were collected and treated (split-sample) as BioXcell with 4 h equilibration and either Triladyl or OptiXcell, both with 24 h equilibration. A total of 11,059 straws were used for insemination of cows and heifers. For Triladyl, progressive sperm motility, acrosome defects, and plasma membrane and acrosome integrity improved with a 24 h compared to a 4 h equilibration time. Four bulls each were used with Triladyl and OptiXcell for trial 2. In trial 2, non-return rates did not differ among groups. Therefore, using a 24 h equilibration time might improve in vitro sperm parameters, depending on the extender used. Moreover, it would be possible to change from 4 h to 24 h equilibration time without impairing field fertility.

Improvement of post-thaw quality and fertilizing ability of bull spermatozoa using Rho kinase inhibitor in freezing extender.

In this study, it was hypothesized that the addition of an appropriate concentration of Y-27632 (a ROCK inhibitor) to the freezing extender prevents cryopreservation-induced apoptosis and improves embryonic development after in vitro fertilization (IVF). Semen samples were collected from five fertile Simmental bulls using an artificial vagina twice a week for 4 weeks. Selected samples were pooled and diluted with Tris-egg-yolk-glycerol (TEYG) extender containing different concentrations of Y-27632 (0, 10, 20, 30, and 40 μM) and then frozen in liquid nitrogen. After thawing, computer-assisted semen analysis (CASA), plasma membrane integrity, and acrosome intactness were evaluated in terms of morphological abnormalities, intracellular generation of reactive oxygen species (ROS), DNA fragmentation, phosphatidylserine (PS) externalization, and apoptotic-related gene expression. Finally, groups of frozen and thawed spermatozoa were used for bovine oocyte IVF. The results show that the semen extender at a concentration of 20 μM Y-27632 effectively improved total motility (TM), curvilinear velocity (VCL), as well as the plasma membrane and acrosome integrity compared to the control group (p < 0.05). Intracellular ROS levels were significantly (p < 0.05) lower in samples treated with 30 μM Y-27632 compared to the control specimen. Furthermore, supplementation of the semen extender with 20 μM Y-27632 resulted in more viable spermatozoa compared with the control group (p < 0.05). According to qRT-PCR results, the expression levels of BAX and CASPASE-9 genes in samples treated with 30 μM Y-27632 were significantly downregulated, while the expression of BCL2 was increased compared to the control (p < 0.05). The results of IVF demonstrated that the treatment of frozen-thawed spermatozoa with 20 μM Y-27632 increased blastocyst rates compared to the control group (p < 0.05). In conclusion, the addition of 20 μM Y-27632 into the freezing extender can improve the functionality and the fertilizing capacity of frozen spermatozoa due to its antioxidative and anti-apoptotic properties.

Proton pump inhibitors affect sperm parameters by regulating aquaporins

Proton pump inhibitors (PPIs) were one of the most commonly used drugs in daily life. The adverse effects of long-term use of PPIs have aroused widespread controversy. It was of great significance to explore the molecular mechanism of sperm abnormality caused by PPIs. The PPI group was given omeprazole by gavage for 28 days. After the omeprazole intervention, the caudal epididymis was dissected to obtain sperms, and the sperm was counted through the microscope, as the acrosomal integrity was observed through PNA-FITC staining. The expression of aquaporins were detected by immunofluorescence and western blot in the testis, epididymis and spermatozoa. The liver cytochrome enzyme was evaluated by immunohistochemistry and western blot. We detected the serum estrogen level by ELISA, and the level of alanine transaminase (ALT) were detected through microplate method. The sperm count in PPI group was less than control group (p < 0.05), and the sperm acrosin integrity in PPI group was lower than control group (p < 0.05). In the testis, the expression of aquaporin 3 and aquaporin 8 in PPI group was higher than control group (p < 0.05), while the expression of aquaporin 7 was lower than control group (p < 0.05). In the epididymal and sperm, the expression of aquaporin 3 and aquaporin 7 in PPI group was higher than control group (p < 0.05), while the expression of aquaporin 8 in PPI group was lower than control group (p < 0.05). Meanwhile, the liver cytochrome enzyme in PPI group were lower than control group (p < 0.05), and estrogen and ALT in PPI group were higher than control group (p < 0.05). PPI may lead to the up-regulation of estrogen by inhibiting the activity of cytochrome enzyme, and then lead to the dysfunction of sperm parameters and acrosin integrity by affecting aquaporins function.

Effects of propolis-loaded nanoliposomes fortification in extender on buffalo semen cryopreservation

Buffalo sperm is sensitive to cryoinjuries, thus improving sperm cryoresistance is a critical approach for wide spreading the assisted reproductive technologies in buffalo. The intention of this work was to assess the effect of propolis-loaded in nanoliposomes (PRNL) supplementation of semen extender on semen quality, antioxidant status and some apoptotic genes of cryopreserved buffalo semen. PRNL were prepared using cholesterol (Chol) as well as soybean lecithin and their physicochemical properties were characterized. Egyptian buffalo bulls (4–6 years) were involved, and the semen samples were collected using the artificial vagina method. Buffalo semen was pooled (n = 25 ejaculates) and cryopreserved in tris extender containing PRNL at 0 (PRNL0), 2 (PRNL2), 4 (PRNL4) and 6 µg/mL (PRNL6), respectively. The PRNL had a size of 113.13 nm and a negative zeta potential (− 56.83 mV). Sperm progressive motility, viability, membrane integrity, abnormalities, chromatin damage, redox status, apoptosis status, and apoptotic genes were investigated after post-thawed buffalo semen. Using 2 or 4 µg/mL PRNL significantly increased sperm progressive motility, viability, and membrane integrity, while sperm abnormalities and the percentage of chromatin damages were the lowest in PRNL2 group. Moreover, the PRNL2 group exhibited the best results for all antioxidative activities (TAC, SOD, GPx and CAT) with significantly higher levels than the other groups (P < 0.05). The levels of ROS and MDA were significantly lower in the PRLN2 compared with other groups. The sperm caspase 3 enzyme activities showed the lowest values in PRNL2 groups followed by PRNL4 and PRNL6 groups with significant differences compared with the control. Adding 2 µg/mL PRNL to freezing media significantly reduced apoptotic genes such as Bax and Caspase 3 in sperm, while significantly increase in Bcl2 expression compared with the control (P < 0.001). The expression of Bcl2, Caspase 3 and Bax genes in sperm were not affected by the 6 µg/mL PRNL addition (P > 0.05). The electron micrography descriptions exemplified that the fortification of 2 or 4 µg/mL PRNL maintained the acrosomal and plasma membrane integrities as well as sustained the ultrastructure integrity of the cryopreserved buffalo spermatozoa when compared with control group, whereas the 6 µg/mL of PRNL demonstrated highest injury to the acrosome and plasma membranes. Results show supplementation of the buffalo freezing extender with 2 or 4 µg/mL of PRNL enhanced post-thawed sperm quality via boosting the antioxidant indices, diminishing the oxidative stress and apoptosis as well as maintained the ultrastructure integrity of frozen-thawed buffalo sperm.

Identification of heat shock protein70-2 and protamine-1 mRNA, proteins, and analyses of their association with fertility using frozen-thawed sperm in Madura bulls.

This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

Effect of pentoxifylline on the testicular hemodynamic, volume, testosterone, nitric oxide levels and semen quality in Ossimi rams during non breeding-season

During the non-breeding season, Ossimi rams have testicular regression, including reductions in blood flow, size and spermatogenesis. The objective was to determine the effect of pentoxifylline (PTX) on Ossimi rams during the non-breeding season. Fifteen sexually mature Ossimi rams were allotted to three groups: (1) G0 (n = 5) control group (basic diet, no PTX); (2) G1 (n = 5) 10 mg/kg BW PTX; and (3) G2 (n = 5) 20 mg/kg BW PTX. The PTX was given orally once daily for 7 weeks (wk1 to wk7), whereas ultrasonographic assessment of testes, and collection of semen and blood started 1 week before PTX and were done weekly for 8 weeks (wk0 to wk7). In G2, there was a decrease(P < 0.05) in both Doppler indices (resistive index, pulsatility index) in G2 from wk2 to wk4 and an increase(P < 0.05) in ultrasonographic testicular coloration from wk2 to wk7. Moreover, G2 had the highest (P < 0.05) testicular volume (wk5 to wk7), individual motility, sperm viability and acrosome integrity (wk4 to wk7) and sperm cell concentration (wk6 and wk7). Blood concentrations of testosterone and nitric oxide were increased (P < 0.05) concurrent with decreased Doppler indices. In conclusion, PTX enhanced testicular blood flow and volume, semen quality, and concentrations of testosterone and nitric oxide potential in Ossimi rams during the non-breeding season, with potential to ameliorate deleterious effects of heat stress and perhaps enhance ram fertility.

P-178 The interaction between spermatozoa and cumulus cells: a more physiological approach to the selection of good quality spermatozoa for assisted reproduction

Abstract Study question Does selection by cumulus cells (CCs) improve sperm quality in terms of acrosome integrity and mitochondrial functionality compared to standard techniques? Summary answer The sperm-CCs interaction method appears to be effective in selecting sperm with an intact acrosome and functioning mitochondria What is known already In nature, the selection of the best quality sperm is very demanding and involves high morphological qualities and dynamic features. Semen preparation in ART has long been carried out using either the swim-up or density gradient method; although both methods provide motile sperm, they cannot really replicate the physiological selection processes. As a result, several methods have been proposed to mimic the natural selection that occurs in the female reproductive tract, allowing the most competent sperm to provide the paternal contribution to the zygote. However, the literature on these techniques is limited and does not provide consistent conclusions Study design, size, duration Proof-of-principle study conducted at the New Fertility Group (NFG) in Rome and the Department of Molecular and Developmental Medicine at the University of Siena (Italy) from July 2022 to December 2022, including 9 semen samples. Comparison of the CCs selection model with the standard swim-up by assessing mitochondrial and acrosome integrity. In addition, mitochondrial activity was assessed by oxygraphic analysis in spermatozoa exposed to immortalised human granulosa cells (hGL5) compared to standard swim-up sorted spermatozoa Participants/materials, setting, methods The CCs-model was prepared by running two channels of medium, one with CCs (study group) and one with medium alone (control). Spermatozoa were added to each channel and incubated at 37 °C for 1 hour. Moreover, spermatozoa were also added on hGL5 and incubated for 1.5h. The collected spermatozoa were compared with controls for acrosomal integrity, assessed by FITC-labelled PSA, and mitochondrial membrane potential, assessed by Mitotracker. Finally, sperm oxygen uptake was measured by oxygraphic analysis Main results and the role of chance Assessment of sperm acrosome integrity showed a higher percentage of responding acrosomes in the CCs model compared to swim-up and controls (61% ± 0.3; 39% ± 0.4; 19% ± 0.4 p &amp;lt; 0.05). The CCs model also showed 83% ± 0.3 of spermatozoa with active mitochondria compared to 65% ± 0.3 in the control group (p &amp;lt; 0.005). A similar pattern was observed in the swim-up group. Quantitative analysis of mitochondrial membrane potential (MMP) confirmed that CCs and swim-up groups had higher MMP compared to the control group (p &amp;lt; 0.005). Evaluation of mitochondria in the hGL5 treated group showed active mitochondria in 78% ± 0.49 of the sperm compared to 59% ± 0.65 in the control group (p = 0.1). The same profile was detected by fluorescence quantification of MMP in the hGL5 group compared to the control group (p &amp;lt; 0.01). Oxygraphic analysis, as an index of oxidative phosphorylation and consequently of ATP production, was performed on hGL5 exposed spermatozoa, swim-up selected spermatozoa and controls. hGL5 exposed and swim-up selected spermatozoa showed a similar O2 consumption, about 4 times higher than unselected spermatozoa (p &amp;lt; 0.05) Limitations, reasons for caution The limited sample size and the heterogeneity of the samples were the main limitations of this study. Although CC and hGL5 sperm selection models appear to be more physiological, further studies on other parameters such as sperm DNA integrity are needed. Wider implications of the findings Both the CCs model and the hGL5 model appear to be effective in selecting good quality sperm. As these models avoid sperm centrifugation, further research is needed to understand their efficacy and reliability and whether they are a valid alternative to conventional sperm selection methods in terms of IVF outcomes. Trial registration number none

P-084 Oral antioxidant supplementation ameliorates the deleterious effects of plasticizers Bisphenol AF (BPAF) and Dibutyl Phthalate (DBP) on mouse sperm DNA integrity

Abstract Study question Do common chemicals in plastic, BPAF and DBP, negatively impact sperm parameters and DNA integrity, and can oral antioxidant supplementation mitigate their impact? Summary answer Short-term, low-dose exposure to BPAF or DBP did not impact testicular/epididymal histology but caudal epididymal sperm parameters, particularly DNA integrity was significantly compromised. What is known already Plasticizers are among the most commonly produced chemicals worldwide with 90% of people showing detectable levels in their urine. As endocrine disruptors (EDCs), they act as estrogen mimetics but can also induce oxidative stress and form DNA adducts. Numerous studies have linked exposure to EDCs to impaired spermatogenesis, sperm DNA damage and male infertility. Limited studies suggest antioxidants may alleviate the reproductive toxicity of EDCs. This study measured the effects of short-term exposures to two common environmental EDCs (BPAF &amp; DBP) on the post-testicular compartment epididymis and investigate the efficacy of an oral antioxidant supplement to safeguard sperm DNA integrity. Study design, size, duration Adult male CD1 mice were exposed to low doses (50mg/kg) of BPAF or DBP with or without co-administration of antioxidant supplementation (Fertilix®) for 14 days and evaluated after euthanasia. Participants/materials, setting, methods All experiments were performed at the University Clermont Auvergne, Clermont-Ferrand, France. Mice were supplemented with BPAF, DBP or/and Fertilix® in their drinking water. The results of exposure of males were evaluated against controls and included: testicular and epididymal histology, epididymal ROS status via 4-HNE levels in caput protein extracts, general semen parameters: (count, motility, viability, acrosome integrity), sperm nuclear/DNA parameters [decondensation (Toluidine blue stain), fragmentation (TUNEL test), oxidation (8-OHdG residue level)]. Main results and the role of chance The histology of the testis and epididymis was unaffected by the exposure conditions used. An oxidative stress situation was evident by the EDC exposures, demonstrated by increased level of 4-HNE adducts in the epididymal caput protein extracts. Viability, total and progressive motility, and acrosome integrity of caudal epididymal spermatozoa remained unaltered in exposed animals compared with controls. Exposure to BPAF but not DBP significantly decreased sperm count. Sperm nuclear/DNA integrity assessed by the percentages of sperm with decondensed and oxidized nuclei was significantly increased in EDC-exposed animals. Sperm DNA fragmentation did not significantly increase in any group of exposed animals. Co-administration with Fertilix® significantly corrected the defective sperm parameters (nuclear decondensation and DNA base oxidation) with the exception of sperm count which did not return to control levels in BPAF+ Fertilix® treated animals. These data confirm that even at low doses and short-term exposures, epididymal spermatozoa are susceptible to DNA damage induced by environmental ROS-generating EDCs, confirming its association with male infertility. Limitations, reasons for caution The translation of these findings to humans needs to be confirmed, although the nuclear compartment of human sperm is more susceptible to oxidative alterations than that of the mouse. Wider implications of the findings These data demonstrate the susceptibility of post-testicular sperm cells to common environmental chemicals. They also support the use of appropriate antioxidant supplementation as an effective therapeutic choice for individuals with multiple exposures to environmental stressors such as ROS-generating EDCs. Trial registration number not applicable

Phosphocreatine addition to extender enhances the quality and antioxidant capacity of cryopreserved boar sperm.

Boar sperm are less resistant to drastic changes in the external environment during cryopreservation, mainly because their plasma membranes are rich in unsaturated fatty acids but lack cholesterol and are thus susceptible to lipid peroxidation caused by the attack of reactive oxygen species. This study evaluated the effect of adding phosphocreatine to cryopreservation extenders on boar sperm quality and antioxidant capacity. Different concentrations (0, 5.0, 7.5, 10.0 and 12.5 mmol/L) of phosphocreatine were added to the cryopreservation extender. After thawing, sperm were analysed for morphological parameters, kinetic parameters, acrosome integrity, membrane integrity, mitochondrial activity, DNA integrity and antioxidant enzyme activity. The results showed that 10.0 mmol/L phosphocreatine samples enhanced the boar sperm motility, viability, average path velocity, straight-line velocity, curvilinear velocity and beat cross frequency after cryopreservation and reduced the malformation rate compared to the control group (p < .05). The acrosome integrity, membrane integrity, mitochondrial activity and DNA integrity of boar sperm were higher than those of the control group after adding 10.0 mmol/L phosphocreatine to the cryopreservation extender (p < .05). Extenders containing 10.0 mmol/L phosphocreatine maintained high total antioxidant capacity; elevated the activities of catalase, glutathione peroxidase and superoxide dismutase; reduced malondialdehyde and H2 O2 content (p < .05). Therefore, adding phosphocreatine to the extender is potentially beneficial for boar sperm cryopreservation at an optimal 10.0 mmol/L concentration.