Bovine Spermatozoa Research Articles

Pengaruh Lama Penyimpanan Pengencer Tris Kuning Telur Terhadap Motilitas Spermatozoa Sapi Simental di BIB Banyumulek Provinsi Nusa Tenggara Barat

Tris yolk is a diluent that can be used to maintain and protect spermatozoa during storage so that they can be used in the process of artificial insemination. The length of storage greatly affects the motility and viability of spermatozoa in semen. The purpose of this study was to determine the effect of long storage of egg yolk tris diluent on the motility and viability of simental bovine spermatozoa. This research was carried out at the Banyumulek Artificial Insemination Center Laboratory, West Nusa Tenggara Province, from July to Desember 2024. The study of the effect of long storage of egg yolk tris diluent on the motility and viability of simental bovine spermatozoa used a randomized experimental method complete with four treatments and six repeats. Each treatment, namely P1, P2, P3, and P4, was carried out by mixing 0.075 ml of semen and 0.3 ml of egg yolk tris diluent stored for 24, 48, 72, and 96 hours. The data obtained were processed by variety analysis method (ANOVA) and Duncan's further test. The results showed that P1 treatment with a storage period of 24 hours resulted in the highest average motility and viability values of 63.33 ± 6.055 % for motility and 63.50 ± 6.892 % for spermatozoa viability. P4 treatment with a storage period of 96 hours still showed a good mean value with motility values of 40.83 ± 7.360 % and spermatozoa viability of 53.50 ± 12.112 %. Thus, egg yolk tris is suitable for use as a semen thinner for artificial insemination because it is able to maintain the motility and viability of spermatozoa even for a long time of 96 hours.

Sperm Functional Status: A Multiparametric Assessment of the Fertilizing Potential of Bovine Sperm.

Sperm viability is routinely assessed for the quality control of cryopreserved bovine sperm batches but is not usually conclusive regarding their fertilizing potential. In this study, we investigated the fertility predictive value of bull sperm viability in combination with DNA integrity or the functional status of viable sperm. In addition to sperm viability, we flow cytometrically assessed the percentage of sperm with high DNA fragmentation index (%DFI) and the fraction of viable sperm with low intracellular Ca2+ content and functional mitochondria using the Sperm Chromatin Structure Assay and a five-color staining panel in 791 and 733 cryopreserved batches with non-return rate (NRR) records after ≥100 first services, respectively. Using linear mixed-effects models and conditional inference trees, we examined the potential of sperm viability combined with either DNA integrity or the functional status of viable sperm to predict the batch-specific NRR. Batches with a %DFI of ≤6.86% were more likely to have a NRR of >60%, whereas %DFI values of >6.86% were more likely to be associated with a 55-60% or lower NRR. Combining post-thaw viability with the functional status of viable sperm did not reliably predict the NRR of individual batches. Concluding, the incorporation of DNA integrity assessment can considerably improve sperm fertility prognostics.

PKA and NBC expression patterns in cryopreserved bovine spermatozoa after kaempferol treatment

Kaempferol (KAE) is a member of the flavonoid family, which exhibits antioxidant properties. The richest sources of KAE are green leafy vegetables and herbs like kale, spinach or dill. The goal of our study was to evaluate a possible cryoprotective effects of KAE through to gene expression of protein kinase A (PKA) and sodium bicarbonate cotransporter (NBC). For the experiments we used semen samples obtained from 12 adult Holstein bulls. Before cryopreservation, all samples were divided equally and supplemented by different concentrations of KAE (0, 12.5, 25 and 50 µmol/L), except for native control. After that, RNA was isolated, and the purity and quality of RNA was verified nanophotometrically at A260/A280 nm. Next step was a transcription of RNA into cDNA, which was used for two-step qPCR while expression of PKA and NBC was quantified under specific cycling conditions. Data was evaluated by the delta delta Ct method and One-way ANOVA using Tukey’s range test. According to expression patterns, it was observed that 25 µmol/L of KAE significantly preserved (P<0.0001) the PKA compared to cryopreserved control without KAE treatment. In the case of NBC, there was slightly increase of expression in the group treated with 50 µmol/L of KAE but without significant changes. In conclusion, KAE treatment exhibit promising cryoprotective properties of PKA and NBC genes especially in higher concentration 25 and 50 µmol/L, which may successfully preserved proper expression after thawing of cryopreserved bovine spermatozoa.

Treatment with Pterostilbene Ameliorates the Antioxidant Status of Bovine Spermatozoa and Modulates Cell Death Pathways.

Reactive Oxygen Species (ROS) play an important role in sperm physiology. They are required in processes such as capacitation and fertilization. However, the exposure of spermatozoa to ROS generated from internal or external sources may create a potentially detrimental redox imbalance. Antioxidant supplementation in semen is now a rather common approach to protect spermatozoa from oxidative stress (OS) during their handling and/or cryopreservation. Supplementation with pterostilbene, a potent antioxidant, protects spermatozoa from OS and ameliorates their post-thawing characteristics and viability. In the present study, we used freezing/thawing as a model of natural ROS overproduction and investigated the molecular mechanisms modulated by pterostilbene. Specifically, bovine frozen/thawed spermatozoa were incubated with 10 or 25 μM pterostilbene for 60 min. Results have shown that in a dose-independent manner, pterostilbene decreased lipid peroxidation and increased intracellular GSH levels. Moreover, pterostilbene ameliorated energy production, as ATP and AMP/ATP levels were restored, and increased autophagy levels through AMP-activated protein kinase (AMPK) activation, which finally resulted in the inhibition of apoptotic cell death in bovine spermatozoa when exposed to OS. This study sheds light on spermatozoa redox state, the crosstalk between apoptotic and autophagic pathways, and its role in determining the beneficial or detrimental effect of ROS in spermatozoa.

Mapping the proteome landscape of Indian Zebu (Sahiwal) spermatozoa using high-resolution mass spectrometry and in-silico annotation

Proteomic analysis of sperm cells offers significant insights into proteins’ structural, functional, and localization aspects within biological systems. Sahiwal, a native Indian cattle breed, is well known for its disease resistance, calving ease, and resilience to drought. This study addressed the gap in Sahiwal’s comprehensive sperm proteome profiling data. The research involved the global in-silico quantitative high-resolution mass spectrometry-based protein profiling of Indian Zebu sperm, identifying 4651 sperm proteins. Beyond mere identification, the study characterized these proteins at a sub-organellar level to facilitate a better understanding of their functional attributes. Gene Ontology analysis of sperm proteins facilitated the segregation of proteins based on their function, localization, and mode of action. The study revealed that despite the limited number of organelles, sperm cells encapsulate a wide array of crucial proteins, compensating for the deficiency of organelles through the presence of multifunctional proteins. Most identified sperm proteins actively participate in spermatogenesis, motility, acrosome reaction, capacitation, and seminal plasma binding, directly or indirectly. Notably, the results not only present the highest number of identified bovine sperm proteins but also hold the potential to pave the way for empirical research on sperm functionality, egg-sperm interaction, sperm-sex sorting biomarkers, sperm quality, and bull fertility.

Development and optimization of an efficient RNA isolation protocol from bovine (Bos indicus) spermatozoa

Achieving the optimum extraction of RNA from spermatozoal cells is crucial for carrying out effective high-throughput analysis regarding its role in fertility and other reproduction processes in Bos indicus. Nevertheless, semen comprises spermatozoa and several other secretions from the male reproductive system, which as well consist of diverse somatic cell types. Therefore, the elimination of somatic cells guarantees the purity of the sperm RNA. In the present study, we tested five different RNA isolation protocols and evaluated them for their yield and purity using spectrophotometer and polymerase chain reaction. Among the five RNA isolation protocols, the Triazol + RNAeasy plus Kit + TCEP method revealed optimum performance. We successfully achieved isolation of spermatozoal RNA without any spermatozoal DNA contamination from Bos indicus spermatozoa that contains approx. 1000 to 10,000 times less RNA as compared to other mammalian somatic cells. RNA quality was assessed using primers Protamine1 (spermatozoal RNA and spermatozoal DNA), CDH1 (epithelial cell), KIT (germ cell) and PTPRC (leukocytes) designed using primer BLAST where there was no product amplified except Prm1 whose product size was specific for spermatozoal RNA. The results of our investigation on RNA isolation procedures indicate that the inclusion of a disulphide reducing agent (TCEP) is crucial for the process of sperm cell lysis.

Predictive Indicators of Cryotolerance and Fertility in Bovine Sperm: Evaluating Fresh Semen Quality to Improve AI Outcomes With Frozen-Thawed Sperm.

The success of artificial insemination (AI) with frozen-thawed semen in cattle is influenced by both female factors and sperm quality. In terms of sperm quality, prior studies indicate that the ability of frozen-thawed bovine sperm to fertilise an oocyte is dependent on their quality and resilience to cryopreservation. Cryopreservation induces oxidative stress, leading to ultrastructural damage in the sperm. This study aimed to determine whether the quality of fresh semen can identify bulls with good and poor sperm freezability. This difference between fresh and frozen semen from the same bull allows us to predict fertility. Motility and kinetic parameters were assessed using computer-assisted sperm analysis (CASA), while six functional variables were evaluated through flow cytometry, both before and after the freeze-thaw process on the sperm from 13 bulls. Invivo fertility was measured using 90-day non-return rates. The principal component analysis (PCA) of eight sperm variables post-thaw identified one principal component explaining 81.19% of the total variance and classified the bulls into two groups: Poor freezability bulls (progressive motility: 48.12% ± 8.41%; viability: 77.51% ± 7.61%) and good freezability bulls (progressive motility: 58.64% ± 6.64%; viability: 88.12% ± 2.52%). Bulls with higher freezability showed better sperm viability and motility, as well as lower levels of ROS, superoxides and intracellular calcium before cryopreservation that were significantly correlated with higher non-return rates (NRR). The results underscore the importance of assessing the quality and functionality of fresh semen to predict the fertility potential of cryopreserved sperm. This approach can aid in selecting ejaculates with the best potential for successful artificial insemination, ultimately improving reproductive performance in dairy cattle.

Seminal proteoforms from bulls with contrasting semen freezability: a story deciphered by top-down mass spectrometry.

Top-down proteomics was employed to construct proteoform atlas of sperm and seminal plasma (SP) from bulls with low (LF) and high (HF) semen freezability. Sperm and seminal proteins were fractionated by tandem size exclusion chromatography (< 30 kDa) and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry. This approach enabled the identification of 299 SP (from 46 families) and 267 sperm proteoforms (from 139 families). Seventy proteoforms belonging to beta-defensin 10, c-type natriuretic peptide (NPPC), caltrin, seminal ribonuclease, osteopontin, and binder of sperm protein (BSP) 3 families were unique to HF bulls' SP. LF seminal proteins had unique 77 proteoforms, including caltrin, NPPC, osteopontin, BSP3, serpin family A member 5, and β-NGF families. Proteoform families of SP in HF and LF bulls were related to Ca2+ uptake, capacitation, acrosome reaction, sperm protection, fertilization and proteolytic processes. Thirty-three proteoforms of NPPC, caltrin, and cylicin-2 families were upregulated in HF sperm. Twenty-two proteoforms of caltrin, cylicin-2, ATP synthases, and malate dehydrogenase families were among those upregulated in LF sperm. Truncated and acetylated histone H2A and non-truncated and acetylated c-Myc binding protein were prevalent in LF sperm. Cylicin-2 proteoforms were observed in HF and LF sperm, and truncated glyceraldehyde-3-phosphate dehydrogenases, only in HF sperm. In silico analyses indicated the enrichment of mitochondrial metabolic pathways in HF sperm, including fatty acid metabolism and TCA cycle. Our study brings an unprecedented description of the bovine SP and sperm proteoforms. Post-translational processing appears to define the bio-properties of semen proteins and their associations with sperm cryoresistance.

Adsorption and Distribution of Triplex-Hybridizing Bioconjugated Gold Nanoclusters Inside Spermatozoa- A Study Using Confocal Microscopy and Fluorescence Lifetime Imaging.

The study of the interactions between biofunctionalized gold nanoclusters (Au NCs) and spermatozoa is highly relevant to evaluate the potential of Au NCs as imaging probes and transfection agents in reproductive biology. In this work, confocal laser scanning microscopy (CLSM) was used to investigate the distribution of Au NCs bioconjugated with peptide (nuclear localisation sequence, NLS) and oligonucleotide (locked nucleic acid, LNA) ligands in bovine spermatozoa. Fluorescence lifetime imaging (FLIM) was employed to detect changes in the NC's chemical environment. We observed a pronounced regio-selective accumulation of the bioconjugates in spermatozoa with high concentration at the equatorial segment. Furthermore, 3D-CLSM showed successful non-endosomal cellular uptake of the conjugates by intact sperm cells and the distribution of the bioconjugates was found to be influenced by the ligand types. Interestingly, the FLIM data showed differences in lifetime depending on membrane integrity. Furthermore, ligand-dependent changes in lifetime between NC bioconjugates carrying peptide and oligonucleotide ligands were found, probably attributed to specific interactions with sperm cell compartments.

Microplastics are detected in bull epididymal sperm and polystyrene microparticles impair sperm fertilization†.

An increase in global infertility has coincided with the accumulation of microplastics (MPs) in the environment. This trend is particularly troubling because only 10% of male infertility cases can be attributed to identifiable causes, leaving a knowledge gap in our understanding of their underlying factors. To bridge this, it is important to explore the connection between the accumulation of MPs and the observed decline in male fertility. We assessed the presence of microplastics in epididymal sperm from bulls and used it as baseline concentrations for sperm exposure. MPs were detected in all epidydimal sperm (ES) samples, with a mean concentration of 0.37μgmL-1. Next, to investigate the effects of MPs on fertility, bovine sperm was exposed to three different concentrations of a mixture of 1.1, 0.5, and 0.3μm polystyrene (PS) beads: (1) 0.7μgmL-1, blood concentration of PS in cows (bPS); (2) 0.37μgmL-1, based on the concentration of total MPs found in ES (esMP); and (3) 0.026μgmL-1, based on the concentration of PS found in ES (esPS). All sperm samples incubated with PS exhibited reduced motility compared with the control at 0.5h. However, PS exposure did not affect acrosome integrity or induced oxidative stress. Embryos produced from sperm exposed to PS had reduced blastocyst rates, in addition to increased ROS formation and apoptosis. By employing physiological exposure, this research provided evidence of MPs in bovine epididymal sperm and demonstrated the detrimental effect of PS on sperm functionality.

Cryoprotective Potential of Theobromine in the Improvement of the Post-Thaw Quality of Bovine Spermatozoa.

Theobromine (TBR) is a methylxanthine known for its bronchodilatory and stimulatory effects. This research evaluated the vitality, capacitation patterns, oxidative characteristics, microbial profile and expression of capacitation-associated proteins (CatSper1/2, sodium bicarbonate cotransporter [NBC], protein kinases A [PKA] and C [PKC] and adenylate cyclase 10 [ADCY10]) in cryopreserved bovine spermatozoa (n = 30) in the absence (cryopreserved control [CtrlC]) or presence of different TBR concentrations (12.5, 25, and 50 µM) in egg yolk extender. Fresh ejaculate served as a negative control (CtrlN). Significant post-thaw maintenance of the sperm motility, membrane and DNA integrity and mitochondrial activity (p < 0.001) were recorded following the administration of 25 μM and 50 μM TBR, then compared to CtrlC. All groups supplemented with TBR exhibited a significantly lower percentage of prematurely capacitated spermatozoa (p < 0.001) than CtrlC. Significantly decreased levels of global reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals were observed in the presence of 25 μM and 50 μM TBR (p < 0.01). Western blot analysis revealed that supplementation with 50 μM TBR significantly prevented the loss of NBC and ADCY10 (p < 0.01), while all TBR doses stabilized the levels of PKC (p < 0.05 at 50 μM TBR; p < 0.001 at 12.5 μM and 25 μM TBR). In summary, we suggest that TBR is effective in protecting the spermatozoa during the cryopreservation process through its potential to stimulate energy synthesis while preventing ROS overproduction and the loss of proteins involved in the sperm activation process.

Cryopreservation of bovine sperm causes single-strand DNA breaks that are localized in the toroidal regions of chromatin

BackgroundSperm cryopreservation is widely used in the cattle industry, as it allows for disassociating the localization of sires and the collection of semen from the timing of artificial insemination. While freeze-thawing is known to impair sperm DNA integrity, whether the damage induced consists of single- (SSB) or double-strand breaks (DSB) has not been determined. In addition, no previous study has addressed if DNA breaks preferentially reside in specific genome regions such as those forming the toroid linker regions, or are rather spread throughout the regions linked to protamines. The main aim of the present work, therefore, was to elucidate the type and localization of the DNA damage generated by cryopreservation and to evaluate its impact on artificial insemination outcomes in cattle.ResultsThe incidence of SSB and DSB was evaluated in 12 ejaculates before and after cryopreservation with the Comet assay, and the localization of the DNA breaks was assessed using pulsed-field gel electrophoresis (PFGE). Before cryopreservation, the incidence of SSB was 10.99% ± 4.62% and involved 20.56% ± 3.04% of sperm cells, whereas these figures significantly (P < 0.0001) increased up to 34.11% ± 3.48% and 53.36% ± 11.00% in frozen-thawed sperm. In contrast, no significant differences in the incidence of DSB were observed (P > 0.990) before and after cryopreservation (before: incidence of 13.91% ± 1.75% of sperm DNA affecting 56.04% ± 12.49% of sperm cells; after: incidence of 13.55% ± 1.55% of sperm DNA involving 53.36% ± 11.00% of sperm cells). Moreover, PFGE revealed that the percentage of sperm DNA fragments whose length was shorter than a toroid (< 31.5 kb) was greater (P < 0.0001) after (27.00% ± 4.26%) than before freeze-thawing (15.57% ± 4.53%). These differences indicated that the DNA breaks induced by cryopreservation affect the regions condensed in protamines, which are structured in toroids. On the other hand, in vivo fertility rates were associated to the incidence of SSB and DSB in frozen-thawed sperm (P = 0.032 and P = 0.005), but not with the size of the DNA fragments resulting from these breaks (P > 0.05).ConclusionCryopreservation of bovine sperm generates single-strand DNA breaks, which are mainly located in protamine-condensed toroidal regions. The incidence of DNA breaks in cryopreserved sperm has an impact on cattle fertility, regardless of the size of generated fragments.

Unraveling the role of sperm retained histones in bull fertility and daughter fertility

During spermatogenesis, a substantial proportion of histones are substituted by protamine to condense the genome within the sperm head. Studies indicate that a minority of histones, typically ranging from 1 to 15 %, persist in mammalian sperm post-substitution. The persistence of histones in the zygote facilitates chromatin accessibility to transcription factors in regions crucial for early embryonic development. Nevertheless, the potential causal relationship between retained histones and fertility phenotypes remains uncertain. This study seeks to investigate this relationship. The results indicate that in mature bovine sperm, regions of DNA associated with fertility that bind to histones are primarily concentrated in promoters and transcription start sites, potentially impacting bull fertility and offspring fertility through the regulation of relevant genes. Furthermore, microRNAs and estradiol/ESR are suggested to be the main regulators of the canonical pathways identified, highlighting the need for additional research to investigate their potential utility as biomarkers.

Genetic Variations in the NRF2 Microsatellite Contribute to the Regulation of Bovine Sperm-Borne Antioxidant Capacity.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor protein-coding gene, considered a master regulator of the cellular stress response. The genetic variations of the NRF2 could influence its transcriptional profile and, subsequently, the stress resilience in all cell types, including sperm cells. Therefore, the sperm-borne antioxidants abundance in association with the genetic variation of a GCC microsatellite located at the 5' upstream region of the NRF2 gene was investigated in young (n = 8) and old (n = 8) Holstein bulls' sperm cells at different seasons. The sperm DNA was sequenced using Sanger sequencing, while- the sperm-borne mRNA analysis was carried out using the synthesized cDNA and qPCR. The data were statistically analyzed using GraphPad Prism 10.0.2 software. The results showed that two bulls had a heterozygous genotype of eight and nine GCC repeats, while biallelic of eight, nine, and fifteen repeats were identified in two, ten, and two bulls, respectively. The computational in silico analysis revealed that the NRF2 upstream sequence with 15, 9, and 8 GCC repeats bound with 725, 709, and 707 DNA-binding transcription factor proteins, respectively. Lower quality of sperm DNA was detected in the spring season compared to other seasons and in young bulls compared to old ones, particularly in the summer and autumn seasons. The mRNA expression analysis revealed that the PRDX1 gene was the abundant transcript among the studied sperm-borne antioxidants and was significantly determined in old bulls' spermatozoa. Moreover, two transcripts of the NRF2 gene and antioxidant (SOD1, CAT, GPX1, TXN1, NQO1) genes displayed differential expression patterns between the age groups across seasons in an antioxidant-dependent manner. The bulls with a heterozygous GCC sequence exhibited elevated sperm-borne mRNA levels of NRF2 and PRDX1 transcripts. Taken together, the findings suggest that the NRF2-GCC microsatellite may contribute to the transcription regulation of NRF2 transcripts and their subsequent downstream antioxidants in bovine sperm cells.

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.

RNA Sequencing of Sperm from Healthy Cattle and Horses Reveals the Presence of a Large Bacterial Population.

RNA molecules within ejaculated sperm can be characterized through whole-transcriptome sequencing, enabling the identification of pivotal transcripts that may influence reproductive success. However, the profiling of sperm transcriptomes through next-generation sequencing has several limitations impairing the identification of functional transcripts. In this study, we explored the nature of the RNA sequences present in the sperm transcriptome of two livestock species, cattle and horses, using RNA sequencing (RNA-seq) technology. Through processing of transcriptomic data derived from bovine and equine sperm cell preparations, low mapping rates to the reference genomes were observed, mainly attributed to the presence of ribosomal RNA and bacteria in sperm samples, which led to a reduced sequencing depth of RNAs of interest. To explore the presence of bacteria, we aligned the unmapped reads to a complete database of bacterial genomes and identified bacteria-associated transcripts which were characterized. This analysis examines the limitations associated with sperm transcriptome profiling by reporting the nature of the RNA sequences among which bacterial RNA was found. These findings can aid researchers in understanding spermatozoal RNA-seq data and pave the way for the identification of molecular markers of sperm performance.

Impact of sperm sex sorting on sperm quality and in vitro embryo production in bovine

Increasing evidence suggests that environmental exposures can modify epigenetic marks in the germline, leading to the transmission of abnormal post-fertilization sperm epigenetic indicators and affecting embryonic development. Given the pivotal role of sperm cells in determining embryo quality, there is growing interest in understanding the potential effects of sperm sex sorting on embryo quality. This study aimed to investigate the impact of bovine sperm sexing on in vitro embryo production (IVP) and to associate molecular aspects of embryos analysis. Frozen semen samples from five Nellore bulls were used, with each bull contributing unsexed sperm (conventional semen – CV treatment) and female and male sexed sperm pooled after thawing (SX treatment). First, semen quality was assessed, including motility, morphology, acrosome integrity, and chromatin integrity to denaturation. Then, IVP was carried out, focusing on embryonic production and developmental kinetics. In the third experiment, embryo quality was evaluated by examining the gene expression of key markers (OCT4, NANOG, DNMT3A, TET1, and Fematrin-1) and the methylation pattern of the Satellite-1 and α-Satellite genes in blastocysts. Differences between CV and SX semen were only observed in motility, which was lower in SX compared with CV (P < 0.05). Although cleavage was similar, the SX groups showed lower blastocyst production than CV (P < 0.05). Of the genes evaluated, only NANOG showed high expression in the CV blastocysts compared with the SX blastocysts, but the methylation pattern revealed no differences. In conclusion, sex sorting markedly affects sperm motility and in vitro embryo production but showed no significant impact on embryo quality.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 3: Compositional and molecular changes to the plasma membrane

The aim of this study was to assess the level of membrane cryodamage through the levels of selected capacitation and apoptosis-associated proteins, together with compositional membrane changes in capacitated (CAP), cryopreserved (CRYO) and non-capacitated bovine spermatozoa (CRTL). Sperm kinetic parameters were analyzed by the computer assisted sperm analysis (CASA) while the capacitation patterns were examined with the chlortetracycline (CTC) assay. In the case of DNA integrity, sperm chromatin structure assay and aniline blue staining were used. For the quantification of fatty acid content gas chromatography was performed. Using Western blotting the expression of capacitation (protein kinase C – PKC; phospholipases A2 and Cζ – PLA2, PLCζ; soluble adenylyl cyclase 10 – sAC10) and apoptosis-associated (apoptosis regulator Bax; B-cell lymphoma 2 – Bcl-2; caspase 3) proteins were evaluated. Data indicate a significant decline (p < 0.0001) of sperm kinetic parameters and higher occurrence (p < 0.0001) of DNA fragmentation in the CRYO group. CTC assay revealed a significant increase of acrosome-reacted spermatozoa in the CRYO group when compared to others. Compositional changes in the sperm membrane were visible as a notable decline of docosahexaenoic acid (p < 0.0001) associated with a significant decrease of membrane cholesterol (p < 0.05) and proteins (p < 0.0001) in the CRYO group while the amount of palmitic, stearic, oleic, and linoleic acid increased (p < 0.0001) significantly. Protein expression of all capacitation-associated proteins (PKC, PLA2, PLCζ, sAC10) was significantly down-regulated (p < 0.001; p < 0.0001) in the CRYO group. Relative quantification of apoptosis-associated proteins revealed increased Bax and decreased Bcl-2 levels in the CRYO group, except for caspase-3, which remained without significant changes.

Structural and network analysis of ADAM9 and ADAM10 and its transcript expression in the male reproductive tract of Bos Taurus

A disintegrin and metalloproteinases (ADAMs) are a family of proteins expressed as transmembrane and secreted proteins in tissues. This study investigated the expression of glycoproteins and transcripts of ADAM9 and ADAM10 in different regions of the bovine male reproductive tract (MRT). Fresh bovine samples were collected from the testis, epididymis, and vas deferens of MRT for this study. Special stains were utilized for identifying glycoproteins depending on the type of mucopolysaccharides in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. Quantitative real-time PCR (qPCR) was used to assess the mRNA concentrations of ADAMs9 and 10. Additionally, structural and network analysis was performed to investigate the similarity between human and bovine ADAM9 and ADAM10. Variable glycoprotein (acidic and neutral mucopolysaccharides) expression was observed in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. ADAM9 and 10 mRNA were detected in the testis epididymis and vas deferens, with ADAM9 expression being significantly higher than ADAM10 mRNA expression in the testis and tail of epididymis. Significant similarity between the protein sequence of human and bovine ADAM9 (85.75%) and ADAM10 (96.66%) was observed, while the similarity between protein sequence of ADAM 9 and ADAM10 in humans and bovine was &lt;26%. Bovine ADAM9 showed 4 binding sites, while bovine ADAM10, human ADAM9, and human ADAM10 showed 7 binding pockets, each with considerable variations in the affinity of specific antibody sequences to these binding pockets. Network analysis confirmed that ADAM9 and ADAM10 have critical roles in physiological processes involving cell migration, protease activity, and secretase-related functions. In addition, they have large numbers of hydrogen bonds to most other members of the ADAM family. Our study reports similarities in the expression of glycoproteins and ADAM9 and ADAM10 mRNA in the MRT of Bos Taurus. Structural and network analysis of ADAM9 and ADAM10 suggested functional similarities in these proteins, which are likely to be in facilitating spermatogenesis, transcervical sperm migration, egg-sperm fusion, and fertilization. Further, the similarity between human and bovine ADAM9 and ADAM10 proteins suggests that bovine sperms can be considered as a model system for comparative medicine studies.

The quality and characteristics of bovine sperm are compromised by Toxoplasma gondii antigens, impacting in in vitro bull fertility

Studies in various species have demonstrated different results on the effects of T. gondii infection on sperm quality. It has also been demonstrated that in some stages of the disease, there is elimination of cellular debris or even the intact parasite in the semen. The present work aimed to evaluate the in vitro effects of the presence of soluble T. gondii antigens in bovine semen on sperm integrity. The spermatozoa were treated with T. gondii antigens in double serial dilutions classified as high, medium and low doses (8, 4, 2 µg/ml) in "TALP-Sperm” and “TALP-Fert" media. The results showed that T. gondii antigens affect sperm motility and mitochondrial activity, and cause changes in sperm chromatin integrity, as well as damage to the sperm membrane and acrosome. Finally, spermatozoa treated with T. gondii antigens were evaluated in the in vitro production of embryos (IVEP). The use of semen contaminated with antigens in IVEP routines did not lead to a decrease in the fertilization of oocytes, as sperm undergo selection before in vitro fertilization, which eliminates the most altered sperm. However, early embryonic development was affected, probably by structural changes that were not eliminated in the selection process. The results demonstrated that the presence of soluble T. gondii antigens in bovine semen alters sperm integrity and vital characteristics for the fertilization process and embryonic development and therefore causes fertility problems in males.