Fertilization Ability Research Articles

Adaptive Significance of Long Mating With Repeated Intromissions in Zygogramma bicolorata

ABSTRACTLong matings are abundant in insects despite the range of the costs involved. The causes and consequences of the evolution of long mating remain an interesting problem for behavioural ecologists. We studied extraordinarily long mating that involves repeated intromissions interspersed with latent periods in the Parthenium beetle (Zygogramma bicolorata). We conducted a series of interrupted mating assays to examine the fitness consequences of different components of this curious mating behaviour. We tested multiple adaptive hypotheses concerning male fertility and competitive ability. We found that sperm transfer and fertility did not exhibit a linear increase with the number of intromissions. There was also no evidence of nutrient transfer by the males. Interestingly, our results showed that both sexes suffered a significant cost of long mating. Further, female remating behaviour was found to be modulated by the length of the previous mating. Additionally, males were observed performing a curious leg rubbing behaviour during the inter‐intromission latent period, putatively serving as a copulatory courtship function that reduced female resistance to the continuance of mating. Therefore, we show that while the long mating may still serve a mate‐guarding role, there are additional fitness effects of such behaviour that need careful consideration. Our study provides insights into the adaptive significance of long mating and its fitness consequences.

In vivo fertilization improved the cryotolerance and developmental ability of vitrified-warmed rat fertilized oocytes

The cryopreservation of rat embryos is useful for efficiently archiving rat resources in bioresource repositories. The cryopreserved fertilized oocytes can be quickly reanimated to rats with homozygous mutations using embryo transfer. In addition, cryopreserved rat fertilized oocytes are easier to transport than live animals. Before cryopreservation, fertilized oocytes are obtained by mating or in vitro fertilization. However, it is not clear which fertilized oocytes are most suited to cryopreservation. In this study, we performed a systematic comparison of the fertilizing ability, cryotolerance, and developmental ability of cryopreserved fertilized oocytes at the pronuclear stage produced either by mating (in vivo) or in vitro fertilization (in vitro) in SD and F344 rats. In vivo-fertilized oocytes had higher cryotolerance and developmental ability than in vitro-fertilized oocytes in SD and F344 rats. Furthermore, the fertilization ability, cryotolerance, and developmental ability of vitrified-warmed fertilized oocytes differed between SD and F344 rats. In conclusion, our study suggests that in vivo-fertilized rat oocytes were more suitable for cryopreservation. Our protocol provides an optimized system for the management of rat colonies using fertilized oocytes cryopreservation and contributes to the 3Rs principle by reducing the number of animals used for research.

Effects of nano-berberine and berberine loaded on green synthesized selenium nanoparticles on cryopreservation and in vitro fertilization of goat sperm

After cryopreservation, reactive oxygen species (ROS) can damage sperm. Antioxidants are the primary defense against oxidative damage. Berberine is a bioactive alkaloid found in Berberis vulgaris, Curcuma longa, and Ergon grape, and is a potent antioxidant. Due to the negative effects of free radicals in oxidative stress processes, antioxidant chemicals are required to protect sperm. However, berberine has low bioavailability, making it less effective. Loading techniques on nanoparticles and nanotechnology can help overcome this limitation. Selenium nanoparticles were synthesized with barberry extract, and berberine was loaded on them. Berberine nanoparticles were then synthesized using anti-solvent precipitation with a syringe pump technique. The synthesis of nanoparticles was confirmed by EDX, UV-visible, FE-SEM, Zeta-Potential, and FTIR tests. In this experiment, we aim to investigate the impact of nano-berberine and berberine loaded on Se-NPs on goat sperm parameters after freeze-thawing. We assessed the generation of reactive oxygen species (ROS), in vitro fertility, and the subsequent embryo development of zygote with treated sperm after determining the optimal concentration of various chemicals on sperm parameters. The study found that all treatments had significant differences from the control group in terms of motility, viability, DNA and membrane integrity, ROS level, lipid peroxidation, in vitro fertility ability, and the capacity to develop inseminated oocytes (p < 0.05). The most significant outcomes were observed with berberine loaded on Se-NPs and the combination of selenium nanoparticles with berberine nanoparticles.

GPX5-Enriched Exosomes Improve Sperm Quality and Fertilization Ability

Semen preservation quality affects the artificial insemination success rate, and seminal exosomes are rich in various proteins that are transferable to sperm and conducive to sperm-function preservation during storage. However, the specific effects of these proteins remain unclear. In this study, the specific effects of these proteins on semen preservation quality and fertilization capacity were investigated through a proteomic analysis of seminal exosomes from boars with high conception rates (HCRs) and low conception rates (LCRs). The results revealed significant differences in the expression of 161 proteins between the two groups, with the GPX5 level being significantly higher in the HCR group (p &lt; 0.05). The role of GPX5 was further investigated by constructing engineered exosomes enriched with GPX5 (Exo-GPX5), which could successfully transfer GPX5 to sperm. Compared to the control group, Exo-GPX5 could significantly improve sperm motility on storage days 4 and 5 and enhance the acrosome integrity on day 5 (p &lt; 0.05). Additionally, Exo-GPX5 increased the total antioxidant capacity (T-AOC) of sperm, reduced the malondialdehyde (MDA) level, and decreased the expression of antioxidant proteins SOD1 and CAT (p &lt; 0.05). In simulated fertilization experiments, Exo-GPX5-treated sperm exhibited higher capacitation ability and a significant increase in the acrosome reaction rate (p &lt; 0.05). Overall, Exo-GPX5 can improve boar semen quality under 17 °C storage conditions and enhance sperm fertilization capacity.

Combining insitu rainwater harvesting and integrated nutrient management of organic manure improves soil moisture, fertility and crop yields in marginalized areas: A review

Soil moisture stress and infertility are major biophysical constraints which limit crop production and food security in marginalized areas. Marginal areas are mainly associated with low rainfall which is insufficient to support crops until maturity is reached. The use of insitu rainwater harvesting methods such as planting pits, tied ridges, mulching, Zai pits and half-moon can be a solution to reduce moisture stress. This alone cannot improve crop yields hence the need to use integrated nutrient management (INM) of animal manure, biofertilisers, mineral fertiliser and composts to improve soil fertility, structure and increase water retention ability. Combining rainwater harvesting with INM has the potential to mitigate effects of climate change, improve crop yields and meet food demand in marginal areas. Various nutrient sources used as INM can restore soil health, increase microbial population, nutrient mineralisation and improve soil quality. Use of biofertilisers in agriculture reduces soil toxicity, improve nutrient availability and create a conducive environment for crop growth and development. Integration of tied ridges with cattle manure and mineral fertiliser has the potential of increasing infiltration rates, reducing surface runoff and increase crop yields with 50-150% regardless of soil type. This chapter sought to come up review the effects of rainwater harvesting methods and INM on soil fertility, moisture stress and crop yields in marginalized areas.

Effect of Urolithin A on Bovine Sperm Capacitation and In Vitro Fertilization.

Reactive oxygen species (ROS) play a critical role in the functional competence of sperm cells. Conversely, excessive generation of ROS can impair sperm function, including their fertilization ability. Urolithin A (UA), a gut bacteria-derived metabolite produced from the transformation of ellagitannins, with anti-aging and antioxidant properties, was investigated for the first time in bovine sperm cells in the present study. Firstly, different doses of UA (0, 1, and 10 μM; 8-16 sessions) were used during the capacitation process of frozen-thawed bovine sperm. Sperm motility was assessed using optical microscopy and CASA. Sperm vitality (eosin-nigrosin), ROS, and ATP levels, as well as mitochondrial membrane potential (JC1) and oxygen consumption were evaluated. A second experiment to test the effect of different doses of UA (0, 1, and 10 μM; 9 sessions) in both the capacitation medium, as above, and the fertilization medium, was also implemented. The embryonic development and quality were evaluated. UA, at a concentration of 1 μM, significantly improved sperm movement quality (p < 0.03). There was a trend towards an increase in the oxygen consumption rate (OCR) of capacitated sperm with 1 μM and 10 μM UA supplementation. Moreover, an increase in ATP levels (p < 0.01) was observed, accompanied by a reduction in ROS levels at the higher UA concentration. These results suggest that UA may enhance spermatozoa mitochondrial function, modifying their metabolic activity while reducing the oxidative stress. Also, the number of produced embryos appears to be positively affected by UA supplementation, although differences between the bulls may have mitigated this effect. In conclusion, presented results further support previous findings indicating the potential therapeutic value of UA for addressing reproductive sub/infertility problems and improving ART outcomes. In addition, our results also reinforce the important bull effect on ART and that male sperm bioenergetic parameters should be used to predict spermatozoa functionality and developmental potential.

Antimony exposure affects oocyte quality and early embryo development via excessive mitochondrial oxidation and dysfunction

Antimony (Sb) is a metalloid, widely presents in the environment and associates with human health. In this study, we aimed to decipher whether Sb exposure is harmful to female reproduction and explore the underlying mechanisms. The ICR mice were exposed to 0, 5, 10, and 20 mg/kg acetate potassium Sb tartrate trihydrate by intraperitoneal injection for 10 days, then mouse oocytes were collected for further analysis. We first found a significant decrease in the proportion of MII-stage oocytes obtained from supernumerary ovulation in the fallopian tubes and early embryo development under Sb treatment. Then a series of tests showed Sb affects oocyte maturation by damaging the cytoskeleton of microtubule and actin. Moreover, the abnormal distribution of cortical granules and their component Ovastacin in oocytes, combined with reduced expression levels of Juno, affected sperm-oocyte binding and led to fertilization failure. Based on the sequencing results and experimental validation, it was demonstrated that Sb exposure impairs mitochondrial distribution and membrane potential, elevated levels of mitochondrial superoxide, finally caused energy supply deficits. Mitochondrial damage in oocytes after Sb exposure results in the excessive oxidative stress and early apoptosis. Taken together, these data suggest that Sb exposure decreases oocyte quality and female fertilization ability by impairing mitochondrial function and redox perturbation.

347 Quantifying phenotypic and genetic variation for cow fertility phenotypes in American Simmentals using total herd reporting data

Abstract Inefficiencies in reproduction are the largest drivers of loss in United States beef production. Nearly all reproductive traits are at least modestly heritable, making genetic selection and improvement possible over the long term. Cow-centric reproductive traits present unique challenges, as they are not expressed in male animals and will not be observed until later in the productive life of a female, if at all. This makes cow-centric fertility traits ideal candidates for selection with Expected Progeny Differences (EPDs). We used a commercially generated dataset from the American Simmental Association (ASA) to explore the phenotypic and genetic variation in these traits. The dataset comprised Total Herd Enrollment (THE) records, which required unbiased reporting of enrollment, calving, and disposal for 303,158 females (132,403 cows and 170,755 heifers). Using the THE data, we calculated three continuous and two discrete traits that are indicators of heifer and cow fertility and rebreeding ability. These traits were: 1) calving date (calving date of the cow relative to the start of the calving season), 2) calving interval (days between calves), 3) first calving interval (calving interval observation between the first and second calving record for a female), 4) heifer pregnancy (did the animal calve as 2-yr-old?), and 5) discrete early calving (did animal calve in the first 30 d of the calving season?). We observed a significant difference in calving date in mature cows that calved in the first 30 d of the calving season of their contemporary group as heifers compared with those that calved later (Table 1). Early calving heifers averaged a nearly 10 d sooner calving date throughout their productive lives compared with those that calved after 30 d as heifers (29.9 d vs. 40.2 d, P-value &amp;lt; 0.01). We used a combined pedigree and genomic approach, implemented in GIBBSF90+, to estimate genetic, environmental, and permanent environmental variance components. We estimated heritabilities of 0.24 (± 0.037215) for heifer pregnancy, 0.06 (± 0.009265) for calving date, 0.07 (± 0.015451) for discrete early calving, 0.04 (± 0.003954) for calving interval, and 0.04 (± 0.009604) for first calving interval. We observed positive phenotypic and genetic correlations between these traits, ranging from 0.01 to 0.96 (Table 2). This work provides an extensive population survey of phenotypic trends in reproductive performance and management in a large seedstock population. The heritable nature of the inspected traits motivates further work in understanding their genetic architectures and how they are affected by heterosis in crossbreeding programs.

High temperature induces motility of zebrafish sperm after short-term storage

Short-term storage on ice of zebrafish (Danio rerio) sperm has been successfully applied in diverse basic experimental research. The optimal and economically efficient short-term storage method to date, however, can only maintain the fertilization ability of the sperm for a few hours. In this study, we aimed to improve zebrafish sperm quality after short-term storage and to test whether this affects the nucleotide cargo of spermatozoa. Zebrafish sperm was diluted with E400 extender (130 mM KCl, 50 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM D-(+)-Glucose and 30 mM HEPES-KOH, pH 7.9, 400 mOsmol/kg) at a dilution ratio 1:20 (v/v) in tubes (500 μL) and stored for 24 h at 0–2 °C under aerobic conditions. Elevating the temperature of sperm suspension at 28 °C for 20 min significantly enhanced sperm motility during activation in both distilled water and Perchec solution with 0.25 % Pluronic F-127. Despite the observed decline in sperm motility during short-term storage, Perchec solution (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8.0, 160 mOsmol/kg) enhanced activation of the aged sperm compared to activation of distilled water. Following 24 h of sperm storage, higher fertilization, and hatching rates were observed when sperm suspension was incubated at 28 °C for 20 min and activated in Perchec solution at a ratio of approximately 6120 spermatozoa per egg, compared to activation in hatchery water. A tentative RNA-seq study identified a set of 2180 differentially expressed genes after 24 h of sperm storage, significantly enriched in the glycosaminoglycan degradation and galactose metabolism pathways. The validity was discussed due to the limited mRNA content in spermatozoa. No alterations were identified in the DNA methylation level of 24 h stored sperm and neither differentially methylated CpGs (DMCs) nor differentially methylated regions (DMRs) were detected between fresh and aged sperm. Based on these phenotypic and molecular investigations and the successful implementation of high-temperature incubation prior to sperm activation in artificial reproduction, we propose that high-temperature incubation following short-term sperm storage at low temperatures is a safe and significant approach in zebrafish and holds potential benefits for other economically important warm-water species in aquaculture.

Higher abundance of DLD protein in buffalo bull spermatozoa causes elevated ROS production leading to early sperm capacitation and reduction in fertilizing ability

BackgroudBefore fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm’s ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction.ResultsIn this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P < 0.05) higher DLD abundances. Herein, we optimised the MICA concentration to inhibit DLD function, spermatozoa were treated with MICA in time (0, 1, 2, 3, 4, and 5 h) and concentrations (1, 2.5, 5, and 10 mmol/L) dependent manner. Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration, which was used for further experimentation in HF and LF. Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly (P < 0.05) higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa. The kinematic parameters of the spermatozoa such as percent total motility, velocity parameters (VCL, VSL, and VAP) and other parameters (BCF, STR, and LIN) were also decreased in MICA treated spermatozoa in comparison to the control (capacitated) spermatozoa.ConclusionsThe present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation, which subsequently reduces their capacity to fertilize.

Incubating frozen-thawed buffalo sperm with olive fruit extracts counteracts thawing-induced oxidative stress and improves semen quality

Freezing-thawing procedures and semen manipulation for in vitro fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress.Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 106/mL in IVF medium with 0, 72, 143, and 214 μL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) μM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes.The treatment with OFE at all concentrations tested increased (P < 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (P < 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (P < 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (P < 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (P < 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (P < 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and in vitro fertilization ability by enhancing sperm antioxidant capacity.

Unveiling the Potential of Aloe vera Gel Supplementation in a Cooling Extender: A Breakthrough in Enhancing Rooster Sperm Quality and Fertility Ability.

The cooling of semen storage at 5 °C from a Thai native rooster (Pradu Hang Dum), supplemented with herbs possessing antioxidant properties, provided limited research. This study was conducted to evaluate the efficiency of Aloe vera (AV) gel supplementation at various levels on the quality of cooled semen and subsequent fertility after artificial insemination. Sixty-four chickens had semen pooled, diluted, and supplemented with different levels of AV gel (0% as control, 0.25%, 0.50%, 1.0%, 2.5%, 5.0%, 10%, and 20%), and then stored for 72 h. In Experiment 1, semen quality, malondialdehyde (MDA) levels, and pH values were assessed at 0, 24, 48, and 72 h after storage. Experiment 2 assessed fertility potential using the most effective cooled storage semen from Experiment 1. Results showed a decrease in semen quality with prolonged storage time (p < 0.001). The highest semen quality was observed in the group supplemented with 1.0% AV gel (p < 0.001), whereas the lowest was noted in the 20% AV gel group (p < 0.001). Furthermore, the 1.0% AV gel group exhibited the highest semen quality at 24, 48, and 72 h of storage. The evaluation of fertility and hatchability rates revealed a statistically significant improvement in fertility potential (p < 0.05) in the group supplemented with 1.0% AV gel. In summary, this study represents the first investigation of stored Thai native rooster semen using a semen extender supplemented with Aloe vera gel at 5 °C, demonstrating its efficacy for storage up to 72 h. The addition of 1% AV gel was recommended as an antioxidant supplementation during the semen storage process at 5 °C to enhance semen quality and fertility rates.

Drosophila melanogaster sperm turn more oxidative in the female.

Males and females of many species store sperm for extended periods. During storage, sperm are predicted to undergo cellular and functional changes, especially towards glycolytic energy metabolism because oxygen radicals derived from oxidative phosphorylation can affect sperm motility and fertilisation ability. However, not all species can use both major energy metabolism pathways. Here, we examined the fruit fly Drosophila melanogaster and asked whether sperm metabolism can be fuelled by both glycolysis and oxidative phosphorylation, and to what extent metabolism changes during storage. Inhibiting glycolysis in vitro led to a more oxidative state of sperm; inhibiting oxidative phosphorylation increased the glycolytic component, assessed by multi-photon autofluorescence lifetime imaging (FLIM) of NAD(P)H. We further examined sperm in male and female sperm storage organs using FLIM of NAD(P)H and FAD. In intact storage organs, we found that, unexpectedly, (i) sperm were more oxidative in females than in males, and (ii) oxidative phosphorylation increased with storage duration in females. Our observation that the relative contribution of the two major energy metabolic pathways in D. melanogaster sperm differs in males and females and over storage time has important evolutionary implications.

Cryoprotectant-specific alterations in the proteome of Siberian sturgeon spermatozoa induced by cryopreservation

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods—liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.

4-methylimidazole exposure impairs sperm mobility by reducing the expression of blood-testis barrier junction protein in mouse testes

4-methylimidazole (4-MI), a derivative of imidazole, is a widely used component in caramel-colored food products such as soy sauce, beer and other soft drinks. The present study is aimed to investigate the effects of 4-MI on the male reproduction. The results revealed that 8 weeks of 4-MI exposure did not significantly alter the body weight and testicular weight of male mice. However, testicular morphology and computer-assisted sperm analysis showed that exposed to 4-MI caused irregular arrangement of spermatogenic cells in the testes and weakened sperm motility. Consistently, we observed the decreased fertilization ability in vivo of 4-MI-treated male mice. We further demonstrated that 4-MI disrupted the blood-testis barrier (BTB) integrity by decreasing the protein expression of BTB-related junction with permeability assay and western blot. In addition, the apoptosis of Sertoli cells (TM4) occurred in 4-MI treated mice, which might be caused by the generation of oxidative stress. Collectively, our findings document that 4-MI exposure damages the sperm mobility via disruption of BTB integrity.

Influence of incorporating L-carnitine or Moringa oleifera leaves extract into semen diluent on cryosurvival and in vitro fertilization competence of buck sperm

This study aimed at scrutinizing efficiency of incorporating L-carnitine or M. oleifera leaves extract into semen diluent on improving cryopreservation capacity and in vitro fertilization ability of buck spermatozoa. Ejaculates (n=48) were collected by an artificial vagina from six adult Damascus bucks twice weekly during the breeding season (September–October). Following initial evaluation, ejaculates of each collection session from the same bucks were pooled, diluted (1:10) with glycerolized (3 % glycerol, v/v) tris-citric acid egg yolk diluent and were split into three aliquots. The first aliquot served as control, whereas the second and third aliquots were supplemented with 4 μL/mL L-carnitine and 400 μL/mL moringa leaves extract (v/v), respectively. Thereafter, all specimens were processed for cryopreservation and were stored in liquid nitrogen (-196 °C) for 12 months before post-thaw sperm criteria were analyzed by a computer-assisted sperm analysis (CASA) system. Integrity of sperm DNA post thawing was visualized in all semen groups by fluorescence imaging, and in vitro fertilization ability of spermatozoa was also determined. Inclusion of L-carnitine or moringa leaves extract into the diluent improved (P<0.05) post-thaw sperm physical, morphofunctional and kinematic attributes, whilst maintaining (P<0.05) integrity of sperm DNA throughout the freezing and thawing cycle. Consequently, both supplemented groups yielded higher (P<0.05) in vitro fertilization rates compared to control. These results accentuate the protective roles of these antioxidants on buck sperm against consequences of cryopreservation-induced oxidative stress, hence ameliorating post-thaw sperm quality and fertilization competence. This is crucial for successful application of AI and IVF in goat selective breeding programs.

CHANGES IN THE METABOLISM OF NATIVE AND DECONSERVED BULL SPERMATOZOA UNDER THE ACTION OF MOLECULAR HYDROGEN

Background. The process of storing sperm in a deeply frozen state causes structural and functional disorders of spermatozoa, which reduces their fertilizing ability. The main mechanism of the negative effect of cryopreservation on spermatozoa is the development of oxidative stress. Molecular hydrogen has some advantages as a potential antioxidant molecule - it is a selective effect on certain reactive oxygen species, the ability to overcome cell membranes, the absence of toxic effects. Purpose. To study the effect of molecular hydrogen on the functional status of native and deconserved sperm cells of breeding bulls. Materials and methods. The object of the study was the ejaculates of bulls before cryopreservation and after defrosting. The native sperm diluted with the "BioXcell" diluent, the native sperm diluted with the "BioXcell" diluent enriched with molecular hydrogen, the sperm after deep freezing and the sperm after deep freezing pretreated with molecular hydrogen were studied. The intensity of free radical processes and the activity of antioxidant enzymes were determined in spermatozoa. Results. After cryopreservation of sperm, the processes of lipid peroxidation in spermatozoa are significantly activated. The content of malondialdehyde, diene and triene conjugates increases. The use of molecular hydrogen to correct the quality of sperm production after cryopreservation gave positive results. A decrease in the concentration of primary and intermediate products of lipid peroxidation was noted. The activity of superoxide dismutase and catalase in spermatozoa significantly increases. Conclusion. Molecular hydrogen has the potential as a new and effective antioxidant, the widespread use of which is possible for veterinary purposes. Sponsorship information. The study was funded by the Russian Science Foundation grant No. 23-26-00205.

Seed priming as a promising technique for sustainable restoration of dryland

The success of plant growth and development in dryland restoration relies on seed germination, seedling development, and establishment. The lack of rainfall and extreme temperature variations in arid regions impede germination and seedling establishment, necessitating new approaches and techniques. The present review summarizes the vital role of seed priming techniques for the sustainable restoration of drylands through their effects on enhancing seed germination, seedling growth and development, plants' nutrient use efficiency, soil fertility, essential enzymes, microbial community, and stress adaptation ability, helping to alleviate the effects of abiotic stresses on plants. The importance of seed priming is discussed in both agricultural and ecological restoration contexts. Priming maximizes crop yields, reduces crop failure, and promotes eco‐friendly farming practices. In ecological restoration, seed priming enhances plant establishment and survival, contributing significantly to the restoration of native vegetation and wildlife habitats. The review categorizes seed priming techniques into six subcategories, highlighting their unique advantages for optimizing restoration efforts. The review highlights the underlying mechanisms of seed priming techniques to improve seed germination and plant development in dryland regions, particularly under salinity and drought constraints. While seed priming has proven effective, it also faces several limitations, such as cost, seed longevity, and environmental concerns. Investigating environmentally friendly priming methods and understanding the prolonged effects of priming on seed viability and the environment is crucial. Further research is needed to find cost‐effective, eco‐friendly seed priming methods, and to explore the genetic and molecular bases of stress tolerance in various plant species across diverse environments.

Broadening the genetic base of the diploids with biotic resistance to accelerate banana (Musa acuminata L.) breeding

Success of banana (Musa acuminata L.) breeding mainly depends on the male diploid parents used in the hybridization program. Developing intermediate diploid breeding lines with fertile pollen production ability, resistance to biotic and abiotic stresses, and fruit quality through the diploid x diploid strategy is the key step in improving commercial cultivars. Till now, at the global level only a limited number of diploid accessions is being used in banana breeding programs. In the present study, through the diploid x diploid breeding strategy, the genetic diversity of diploids has been broadened by developing nine polleniferous biotic-stress-resistant diploid lines. Among these, except for two progenies (P), all were resistant to the eumusae leaf spot disease. A single progeny (P 134) showed resistance to root-lesion and root-knot nematodes, whereas P 148 showed moderate resistance to root-knot nematodes alone. Three progenies of cv. Rose x Pisang Lilin (P 427, P 428 and P 429) were moderately resistant to banana stem weevil. Irrespective of cross combinations, barring two, all other progenies exhibited resistant reactions to fusarium wilt, Foc race 1 under both hot spot and pot culture screening. Among the nine diploid progenies, P 427, P 428 and P 429 were the best prospects to contribute multiple resistance against pests (banana stem weevil) and diseases (eumusae leaf spot and Foc race 1), whereas P 134 could be used as the resistant source for root-knot nematodes. The pyramiding of resistant/defense-related alleles in the progenies has been confirmed through SSR markers associated with Foc resistance. These polleniferous resistant improved diploid progenies are the potential source for accelerating the banana breeding program for improving biotic resistances in the triploid commercial cultivars through reconstructive breeding and/or for stacking the resistant genes in other genetic backgrounds.

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol=120.9±2.9 vs. NC=76.91±6.9µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol=1.15±0.02 vs. NC=0.77±0.03µm) (straightness: bicarbonate and polyvinyl-alcohol=0.76±0.01 vs. NC=0.87±0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.