Bovine Spermatozoa Research Articles

Influence of seminal plasma during different stages of bovine sperm cryopreservation.

This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n=42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma=SP group; without seminal plasma=NSP group) and packed to a final concentration of 50×106 sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p≤.05 was considered significant. A time effect was observed for all sperm characteristics (p<.05), except for chromatin fragmentation (p>.05). The presence of seminal plasma better preserved the acrosomal integrity (SP=75.2% and NSP=71.7%; p<.05) and also provided lower F-actin remodelling during cryopreservation process (SP=29.9% and NSP=32.4%; p<.05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p<.05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (-65.2%), acrosomal membrane (-34.0%) and mitochondrial potential (-48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation.

Fortification of Tris Extender with Mifepristone, Sericin and Taurine Improves Velocity and Kinematics of Fresh and Frozen-thawed Bovine Spermatozoa

Background: The evaluation of bull fertility essentially involves semen analysis and conception rates among inseminated females. The conventional evaluation is relatively imprecise, time consuming and subjected to individual variations, while computer assisted sperm analysis (CASA) is a quite faster, precise and objective tool to identify differences in sperm motility and velocity/kinematics attributes and avoids subjective errors. The present study was planned to evaluate and compare the CASA traits of fresh and frozen-thawed semen of Gir and Murrah bulls in Tris extender without and with different antioxidant additives by adopting Biovis CASA.Methods: The semen ejaculates with greater than 75% initial motility from 3 Gir and 3 Murrah bulls were split-diluted at 100 million sperm per ml using Tris-citrate-fructose-yolk-glycerol (TFYG) extender without (control) and with three antioxidant additives, viz., Mifepriston (10 µg/ml), Sericin (5 mg/ml) and Taurine (4 mg/ml), were filled in French mini straws and frozen in liquid nitrogen vapour using a programmable biofreezer. The freshly diluted and frozen-thawed semen samples were assessed for sperm motion characteristics, velocity/kinematics using Biovis CASA. Result: The mean percentages of total motile spermatozoa, irrespective of additives, in freshly diluted and frozen-thawed semen were 85.50±0.92 and 48.76±1.69 for Gir bulls and 85.03±0.72 and 52.61±1.46 for Murrah bulls, respectively. The mean total motile as well as rapid and slow progressive motile sperm percentage were significantly enhanced by fortification of TFYG extender with Mifepristone than in control extender, while values for Sericin and Taurine were intermediary and statistically similar to Mifepristone. The per cent decline in total motile sperm due to freezing-thawing was more or less same in both the breeds for all the extender additives, being lowest in Mifepristone supplemented extender in cattle (32.27 vs 48.81%) and buffalo (31.27 vs 43.14%) semen. The rapid and slow progressive motile sperm also followed the same pattern. The values of VAP and VCL were significantly (p less than 0.05) higher and VSL lower in Murrah than Gir breed at post-thaw stage. The overall mean linearity (%), straightness (%), beat-cross frequency (hz), lateral head displacement (µm), wobbling index (%), dancing velocity (µm2/s) and dancing mean (µm2/s) of sperm were higher in Murrahs than in Gir semen with significant (p less than 0.01) difference at post-thaw stage. The fortification of extender with Mifepristone improved all these traits at post-thaw stage compared to other additives and control TFYG extender. The per cent decline in these traits due to freezing stress was much lower in buffalo sperm than the cattle sperm particularly with Mifepristone fortification. It was concluded that freezing-thawing stress adversely affected the motion and kinematics of both cattle and buffalo sperm and that Tris extender fortified with antioxidant additives, particularly Mifepristone @ 10 µg/ml, protected sperm from adverse effect of dilution and cryopreservation with improved post-thaw sperm quality.

Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling.

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of SEPT7 siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and SEPT7 siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202-SEPT7 mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202/SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulates the first cleavage process of bovine embryos by SEPT7 and demonstrate the potential of sperm-borne miRNAs to improve the efficiency of SCNT.

Glutationa e IGF-1 na criopreservação seminal bovina: resposta do estresse oxidativo e taxa de gestação

ABSTRACT The objective of this study was to evaluate the rate of conception, metabolic, and structural conditions of cryopreserved bovine sperm cells, plus extender with IGF-1 and glutathione (GSH). 12 ejaculations of Nelore bulls were used, submitted to treatments: control, gSH (2mM/mL), IGF-1 (100ng/mL) and gSH (1mM/mL) + IGF-1 (50ng/mL). After cryopreservation and thawing the semen passed the fast thermo resistance test (TTR), plasma membrane and acrosomal integrity (PIAI), mitochondrial membrane potential (AP), oxidative stress, and conception rate. Tukey test was used for the statistical analysis of the parametric variables and the Friedman test for nonparametric. The gestation percentage was compared by the Chi-square test. There was no statistical difference (P&lt;0.05) between treatments for the TTRr variable. Otherwise in the oxidative stress evaluated with the CellROX probe was noted that the IGF-1 showed the highest number of reactive cells (P&lt;0.05). The PIAI, AP and gestation rate showed no difference among treatments (P&gt;0.05), with an average of conceptions of 36.58%. It is concluded that IGF-1, gSH and their association did not cause changes in sperm motility, mitochondrial potential, plasma and acrosomal membrane integrity. IGF-1 increased oxidative stress, however, there was no difference in the gestation rate among the treatments.

Epidermal growth factor alleviates the negative impact of urea on frozen-thawed bovine sperm, but the subsequent developmental competence is compromised

Upon insemination, sperm cells are exposed to components of the female reproductive tract (FRT) fluids, such as urea and epidermal growth factor (EGF). It has been shown that both urea and EGF use EGF receptor signaling and produce reactive oxygen species (ROS) that are required at certain levels for sperm capacitation and acrosome reaction. We therefore hypothesized that during bovine sperm capacitation, a high level of urea and EGF could interfere with sperm function through overproduction of ROS. High-level urea (40 mg/dl urea is equal to 18.8 mg/dl of blood urea nitrogen) significantly increased ROS production and TUNEL-positive sperm (sperm DNA fragmentation, sDF) percentage, but decreased HOS test score, progressive motility, acrosome reaction and capacitation. The EGF reversed the negative effects of urea on all sperm parameters, with the exception of ROS production and DNA fragmentation, which were higher in urea-EGF-incubated sperm than in control-sperm. The developmental competence of oocytes inseminated with urea-EGF-incubated sperm was significantly reduced compared to the control. A close association of ROS production or sDF with 0-pronuclear and sperm non-capacitation rates was found in the network analysis. In conclusion, EGF enhanced urea-reduced sperm motility; however, it failed to reduce urea-increased sperm ROS or sDF levels and to enhance subsequent oocyte competence. The data suggests that any study to improve sperm quality should be followed by a follow-up assessment of the fertilization outcome.

Impact of Segmented Magnetization on the Flagellar Propulsion of Sperm‐Templated Microrobots

Technical design features for improving the way a passive elastic filament produces propulsive thrust can be understood by analyzing the deformation of sperm‐templated microrobots with segmented magnetization. Magnetic nanoparticles are electrostatically self‐assembled on bovine sperm cells with nonuniform surface charge, producing different categories of sperm‐templated microrobots. Depending on the amount and location of the nanoparticles on each cellular segment, magnetoelastic and viscous forces determine the wave pattern of each category during flagellar motion. Passively propagating waves are induced along the length of these microrobots using external rotating magnetic fields and the resultant wave patterns are measured. The response of the microrobots to the external field reveals distinct flow fields, propulsive thrust, and frequency responses during flagellar propulsion. This work allows predictions for optimizing the design and propulsion of flexible magnetic microrobots with segmented magnetization.

Carryover effect of atrazine and its metabolite-from treated bovine spermatozoa to the embryo's transcriptome†.

Atrazine (ATZ) is an extensively used herbicide and ubiquitous environmental contaminant. ATZ and its metabolite, diaminochlorotriazine (DACT), cause several cellular and functional alterations in spermatozoa. We aimed to examine the effect of ATZ/DACT on spermatozoon DNA integrity, fertilization competence, embryonic development, and transcriptome profile of in vitro-produced embryos derived from fertilization with pre-exposed sperm. Bovine spermatozoa exposed to ATZ (0.1 or 1μM) or DACT (1 or 10μM) during in vitro capacitation were used for in vitro fertilization of untreated oocytes. Cleavage and blastocyst-formation rates were evaluated 42h and 7days postfertilization, respectively. The association between DNA fragmentation and apoptosis (annexin V kit) was determined. Fertilization competence of annexin-positive (AV+) and annexin-negative (AV-) spermatozoa was examined. Microarray analysis was performed for 7-day blastocysts. Intracytoplasmic sperm injection was performed with control (AV+, AV-) and DACT (AV+, AV-) spermatozoa. Cleavage rates did not differ between groups and blastocyst formation tended to be higher for AV- vs. AV+ in both control and DACT groups, suggesting that acrosome reaction, rather than DNA fragmentation, underlies the reduced cleavage. Transcriptomic analysis revealed 139 and 230 differentially expressed genes in blastocysts derived from ATZ- and DACT-exposed spermatozoa, respectively, relative to controls. Proteomic analysis shown differential expression of proteins in ATZ- or DACT-treated spermatozoa, in particular proteins related to cellular processes and biological pathways. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. Overall, the current study reveals a deleterious carryover effect of ATZ/DACT from the spermatozoa to the developing embryo.

Establishment of bioinformatics pipeline for deciphering the biological complexities of fragmented sperm transcriptome

Despite the development of several tools for the analysis of the transcriptome data, non-availability of a standard pipeline for analyzing the low quality and fragmented mRNA samples pose a major challenge to the computational molecular biologist for effective interpretation of the data. Hence the present study aimed to establish a bioinformatics pipeline for analyzing the biologically fragmented sperm RNA. Sperm transcriptome data (2 x 75 PE sequencing) generated from bulls (n = 8) of high-fertile (n = 4) and low-fertile (n = 4) classified based on the fertility rate (41.52 ± 1.07 vs 36.04 ± 1.04%) were analyzed with different bioinformatics tools for alignment, quantitation, and differential gene expression studies. TopHat2 was effectual compared to HISAT2 and STAR for sperm mRNA due to the higher exonic (6% vs 2%) mapping percentage and quantitating the low expressed genes. TopHat2 also had significantly strong correlation with STAR (0.871, p = 0.05) and HISAT2 (0.933, p = 0.01). TopHat2 and Cufflinks combo quantitated the number of genes higher than the other combinations. Among the tools (Cuffdiff, DESeq, DESeq2, edgeR, and limma) used for the differential gene expression analysis, edgeR and limma identified the largest number of significantly differentially expressed genes (p < 0.05) with biological relevance. The concordance analysis concurred that edgeR had an edge over the other tools. It also identified a higher number (9.5%) of fertility-related genes to be differentially expressed between the two groups. The present study established that TopHat2, Cufflinks, and edgeR as a suitable pipeline for the analysis of fragmented mRNA from bovine spermatozoa.

L-amino acid oxidase 1 in sperm is associated with reproductive performance in male mice and bulls

Sperm quality is an important indicator of male fertility, and a suitable biomarker enables the selection of high-quality spermatozoa. We previously found that L-amino acid oxidase encoded by the Lao1 gene, exerts biological roles in the mammary gland and brain by converting specific L-amino acids into keto acids, ammonia, and hydrogen peroxide (H2O2). Here, we describe the role of Lao1 in male reproduction. Lao1-deficient (Lao1-/-) male mice generated fewer pregnant embryos and pups as well as lower ratios of fertilized oocytes even ovulated number was not different, suggesting that male subfertility caused the smaller litters. We found that LAO1 expressed in acrosomes is associated with high malformation ratios and low viability of Lao1-/- sperm. Wildtype (WT) sperm produced more H2O2 than Lao1-/- sperm and 10μM H2O2 restored KO sperm viability in vitro. In addition, the sperm ratio of induced acrosome reaction was higher in WT than in Lao1-/- sperm incubated with the calcium ionophore A23187. Moreover, LAO1 expression was abundant in bovine sperm with high fertilization ratios. We concluded that LAO1 localized in the sperm acrosome influences sperm viability and morphology as well as the acrosome reaction, and that LAO1-deficient sperm might cause male subfertility. Thus, LAO1 might serve as a novel marker for selecting high-quality spermatozoa, especially for livestock reproduction.

Effects of Dietary Supplementation of Conjugated Linoleic Acids and Their Inclusion in Semen Extenders on Bovine Sperm Quality.

Simple SummarySuboptimal fertility in males accounts for about two-thirds of infertility cases, thus being of serious concern for the dairy industry, where optimal fertility is fundamental for farm profitability. Although genetic defects responsible for subfertility have been identified, the role of seminal compounds on fertility remain unclear. Feeding rumen-protected isomers of conjugated linoleic acid (CLA) to dairy cows reportedly enhances circulating insulin-like growth factor I (IGF-I) levels. In breeding bulls, the IGF-I concentration in seminal plasma has been positively correlated with fertility rates. Therefore, the objective of the study was to evaluate the effect of dietary CLA supplementation and of their inclusion to the semen extender on bovine semen quality and freezability.Isomers of conjugated linoleic acid (CLA) enhances circulating insulin-like growth factor I (IGF-I) levels. Furthermore, fertility rate of breeding bulls is positively correlated to seminal plasma IGF-I concentration. Our objective was to evaluate the effect of dietary CLA supplementation and inclusion to the semen extender on bovine semen quality and freezability. Fourteen bulls, randomly assigned to control (CTL) and CLA (50 g/day) groups, were supplemented for 10 weeks. Samples were collected at Weeks −2 (before supplementation), 0, 4, 6 (during supplementation), 10, and 11 (after supplementation). Blood and seminal plasma were analyzed for IGF-I; the ejaculates were frozen in the following subgroups: CTL (no addition to semen extender), CLA c9, t11 (50 µM), CLA c9, t11 (100 µM), CLA t10, c12 (50 µM), CLA t10, c12 (100 µM), and CLA mix (50 µM each of CLA c9, t11 and CLA t10, c12). Sperm motility, morphology, viability, mitochondrial membrane potential, and reactive oxidative species were assessed. CLA supplementation decreased ejaculates’ total volume, increased sperm concentration, beat cross frequency, and decreased oxidative stress; it also increased plasma and seminal plasma IGF-I levels compared to the CTL. The inclusion of CLA c9, t11 100 µM and CLA mixture in the extender increased live spermatozoa percentage post-thawing compared to other groups. Our results show a beneficial effect of CLA supplementation on semen quality; however, further studies evaluating fertilization rates are necessary to corroborate the results.

Experimental study on the effect of flow in microfluidic channel on bovine sperm navigation

The navigation mechanism of mammalian sperm in the female reproductive tract is unclear owing to its complex process. This study performed an in vitro experiment using the microfluidic channel with two reservoirs to investigate the effect of fluid flow on the swimming properties of the bovine sperm. The width and height of the manufactured channel were 200 and 20 μm, respectively. The flow in the microchannel occurs because of the hydraulic head difference between the two reservoirs. Sperm with positive rheotaxis proceed in the opposite direction of the flow in the channel after swimming up the downstream reservoir. This study focused on the effect of the flow in the microfluidic channel on sperm motility. It was observed that sperm mostly moved along the channel wall and accumulated near the wall away from the downstream reservoir. The existence of fluid flow in the channel brought about an increase in the ratio of the sperm with positive rheotaxis. Furthermore, the experimental results indicated that the motility of sperm swimming against the flow along the wall increased away from the downstream reservoir. These results will provide useful information to understand the mechanism of sperm navigation for in vivo fertilization.

Avian sperm increase in vitro the release of exosomes from SST-enriched organoids.

In birds, oviductal cells play a crucial role in the storage of sperm via cell-to-cell communication including extracellular vesicles (EV). We developed a culture of oviductal organoids enriched in sperm storage tubules (SSTorg) to demonstrate the release of EV. SSTorg were cultured for 24 h and added to live (LV), frozen (FZ) and lysed (LY) avian sperm, seminal plasma (SP), avian sperm conditioned medium (CM), or bovine sperm (BV). Western blot demonstrated that SSTorg contained EV protein markers, valosin-containing protein (VCP), heat shock proteins (HSP90AA1, HSPA8), and annexins (ANXA2, A4, A5). Co-culture with LV significantly decreased the intracellular level of all these proteins except HSPA8. Immunohistochemistry confirmed this result for VCP and ANXA4. LY, CM, SP and BV had no effect on the intracellular level of these proteins, whereas FZ induced a decrease in ANXA2, A4 and A5. In culture media, VCP and HSP90AA1 signals were detected in the presence of LV, FZ, BV, LY, CM and SP, but no ANXA4 signal was observed in the presence of FZ and SP. ANXA2 and A5 were only detected in the presence of LV. The most abundant EV were less than 150 nm in diameter. ANXA4 and A5 were more abundant in EV isolated from the SSTorg culture medium. This study provides a useful culture system for studying interactions between SST cells and sperm. We demonstrated the release of EV by SSTorg in vitro, and its regulation by sperm. This may be of crucial importance for sperm during storage in hens.

A mouse seminal vesicle-secreted lysozyme c-like protein modulates sperm capacitation.

Lysozyme (LYZ) c-like proteins are primarily presentin the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c-type LYZ family, the seminal vesicle-secreted LYZ c-like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N-linked glycans. SVLLP is structurally similar to c-type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c-type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin-induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm-oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor.

The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting

BackgroundThe use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRRconv and NRRss, respectively) was recorded for each bull.ResultsIn total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21–5p were significantly correlated to NRRss but not to NRRconv. Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRRss.ConclusionsA set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire’s suitability for the production of sex-sorted sperm.

79 Progesterone-induced acrosome reaction and fertilization rates with bovine intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been a valuable tool in many species because of its ability to overcome male factor infertility problems and eliminate risk of polyspermy; more recently, it been used to improve genome editing technologies. However, limited success with bovine ICSI has hindered these applications in cattle. Numerous treatments have been used to increase the success rate, with marginal improvement. Replicating events synonymous with fertilization, such as the acrosome reaction, may improve fertilization with bovine ICSI. Progesterone (P4) is naturally found in both the cumulus cells surrounding the oocyte and follicular fluid released at ovulation and activates a physiological pathway within sperm to induce an acrosome reaction, a crucial process in fertilization. Progesterone induction of the acrosome reaction as a sperm pretreatment for ICSI has not yet been evaluated in cattle. In this study, frozen–thawed bovine sperm was used. Sperm were first thawed, washed, and separated via gradient to obtain the motile population before capacitation with heparin over 4h before treatment with or without P4 (10 μM) for 15min. To measure the acrosome reacting population, sperm were stained with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) and observed over 2h using cytometric flow analysis and fluorescent microscopy to measure the population undergoing and completing an acrosome reaction. For ICSI, a total of 220 oocytes were used, and both treated and nontreated sperm were incubated for 1h before injection to allow for completion of an acrosome reaction. Fertilization rates were measured by observing pronuclear formation and an absence of sperm 18h after ICSI. Means of acrosome reacting population gathered by flow cytometry and microscopy were analysed by ANOVA. Differences in fertilization rates between groups post ICSI were analysed using a Yates’ corrected chi-squared test. A significantly higher proportion of P4 treated versus control sperm initiated an acrosome reaction at hour 1 (80.2%±4.2 vs. 19%±9.1), which increased to (89.3%±3.9 vs. 29%±8.3) after 2h. P4 also increased the percentage of sperm that completed an acrosome reaction, from 50% (±5.1) at hour 1 and 62.5% (±7.4) at hour 2. Only 14.2% (±3.6) completed acrosome reactions in sperm not treated with P4 by hour 2. Motile sperm in both groups did not decrease over the 2-h incubation time period (P&amp;lt;0.05). Progesterone treatment increased the percentage of fertilized embryos after ICSI, with 38.1% fertilized compared with 10.1% with heparin-treated control injections (P&amp;lt;0.001). These results show that P4 has effects on bovine sperm that allow for higher rates of fertilization after ICSI by utilising the sperm’s physiological response to progesterone. Further embryo development using ICSI with P4-treated sperm, or additional physiologically similar treatments, should continue to be assessed.

80 Energetic substrate availability affects the metabolome profile in bovine sperm

Bovine spermatozoa are specialised cells that require high ATP production for flagellar movement and other physiological events necessary for fertilization. Glycolysis and oxidative phosphorylation (OXPHOS) are the most studied energy pathways in sperm cells and involve metabolites such as glucose, pyruvate, and lactate. Although glycolysis has been described as the preferential pathway for ATP generation in bovine spermatozoa, other metabolites may also be used, leading to distinct metabolome profiles. Thus, the objective of this work was to characterise the metabolome profile of culture media derived from sperm cells incubated in the presence of different energy substrates for ATP production. For that, a semen straw from one bull (n=3) previously tested for IVF was thawed and motile sperm were separated by Percoll gradient, washed and resuspended in FertTalp medium (FT) without capacitator agents (Parrish et al. 1989 Biol. Reprod. 41, 683–699) to a final concentration of 30×106cells mL−1. Then, samples were centrifuged and resuspended in 5 different groups: positive control (PC, FT supplemented with 2mM glucose, 0.2mM pyruvate, and 11mM lactate); negative control (NC, FT without energy substrates); Glu (FT and 3.5mM glucose); Pyr (FT and 0.11mM pyruvate), and Lac (FT and 5.5mM lactate). Samples were incubated at 38.5°C, 5% CO2 in high humidity for 15 and 45min. After incubation, samples were centrifuged and supernatant was collected and analysed by Raman spectroscopy as previously described (Santos et al. 2015 Biomed. Opt. Express 6, 2830–2839; https://doi.org/10.1364/BOE.6.002830). Data were preprocessed and submitted to principal component (PCA) and loading plot analysis (LP) by using Minitab software (Minitab Ltd.). The results showed that after 15min of incubation, the metabolic profiles were similar for the PC, Glu, and Lac groups, suggesting that they present similar metabolic activity. NC and Pyr were a separate cluster, indicating that pyruvate is not metabolized through OXPHOS in this phase. LP analysis comparing Glu and Pyr groups indicated phosphatidylserine, phenylalanine, DNA/RNA, and lipids as the most distinct metabolites. After 45min, PC and Pyr had a similar metabolome profile, whereas NC, Glu, and Lac clustered together, suggesting that for long-period incubation OXPHOS takes place as the preferential pathway for energy production in bovine sperm cells. At this time, the comparison of Glu versus Pyr revealed phosphatidylserine, proline, phenylalanine, carboxylic acid, DNA/RNA, proteins, and lipids as the most different metabolites between groups. Based on these results, we hypothesised that the glycolysis to OXPHOS transition may be a consequence of the depletion of glycolytic enzymes, leading the sperm cells to use distinct pathways for long-term maintenance of ATP production. In conclusion, our data showed that the metabolome profile of bovine spermatozoa varies according to the period of incubation and substrates availability for energy production. However, more studies are necessary to characterise the ability of these metabolites to maintain sperm motility and viability. This research was funded by FAPESP 2017/18384-0.

103 Characterization of the receptors of the endocannabinoid system in equine sperm: Possible role of anandamide in sperm function

Conventional IVF in horses remains challenging. In particular, stallion sperm fails to penetrate the zona pellucida, possibly due to incomplete invitro sperm capacitation. Therefore, there is a need to elucidate, in horses, molecules with a proven role during capacitation in other mammals. Our laboratory has described the relevance of the endocannabinoid system in capacitation of bovine and murine sperm. We reported that anandamide (AEA), an endocannabinoid present in follicular and oviducal fluids, induced capacitation-associated events. The aims of this work were to characterise the localization of cannabinoid receptors in equine sperm and to evaluate the effects of AEA on levels of tyrosine-phosphorylated proteins (pY) and substrates phosphorylated by protein kinase A (pPKA). Both cannabinoid receptors (CB1, CB2, TRPV1) and pPKA and pY were localised in sperm by indirect immunofluorescence. Sperm (15×106mL−1) were incubated, at 38.5°C in air, in modified Tyrode’s-albumin-lactate-pyruvate (TALP) with 25mM NaHCO3, 5mM dextrose and 1mgmL−1 polyvinyl alcohol (PVA; TALP-Bic-PVA) or TALP-Bic-PVA supplemented with AEA (0.1, 1, 10, 100nM, and 1µM) for 4h. After incubation, Western blot was used to determine levels of pY and pPKA in 4.5×106 sperm. Cryopreserved sperm samples from three stallions were evaluated. The normality of data distributions and homoscedasticity were verified with the Shapiro-Wilk and Levene tests, respectively. Data were analysed by one-way ANOVA and Bonferroni post hoc test, with P&amp;lt;0.05 considered significant. Based on immunofluorescence, CB1 was mainly localised in the post-acrosomal region and flagellum (93.4%±5.5, mean±s.d.), CB2 in the post-acrosomal region and middle piece (89.9%±28.3), and TRPV1 in the post-acrosomal region and flagellum (89.3%±9). Sperm positive for pPKA had fluorescence in the middle piece and principal piece of the flagellum. Incubation with 1nM AEA for 4h induced a 61% increase in pPKA levels compared with TALP-Bic-PVA medium alone, with no induction of pY levels in any treatment. In conclusion, cannabinoid receptors were present in equine sperm, and incubation with AEA induced an increase in PKA activity, an essential event associated with sperm capacitation. To our knowledge, this was the first report describing the presence of receptors of the endocannabinoid system in equine sperm and the potential role of AEA in the acquisition of sperm fertilizing ability.

Buserelin Acetate Reduces Mortality and DNA Defragmentation of Bovine Sperm Cells Exposed to Oxidative Stress

Buserelin Acetate Reduces Mortality and DNA Defragmentation of Bovine Sperm Cells Exposed to Oxidative Stress

Semen Sexing in Bovine: Current Status and the Need to Develop Alternative Techniques

Livestock industry is one of the important pillars for country economy. Sperm sexing has the potential of significantly improving the quality and quantity of production in the livestock industries. Over years several attempts, involving several different parameters of sperm cells, have been made to develop an efficient sperm sorting procedure. Till date, the only commercially available method for bovine sperm is flow cytometry. The disadvantages associated with this approach, however, points towards the need for developing a more non-invasive and cost efficient approach for separation of X and Y chromosome bearing spermatozoa. In this review paper, we attempt to highlight all the previously developed approaches, as well as other emerging immunological and proteomics based methods that might have the potential of being developed as a promising breakthrough approach for semen sexing. Furthermore, we discuss the advantages and applications of semen sexing in India and around the world.

Effects of exogenous lactoferrin on characteristics and functions of bovine epididymal, ejaculated and frozen-thawed sperm.

The purpose of this study was to investigate effects of addition of lactoferrin on characteristics and functions of bovine epididymal, ejaculated, and frozen-thawed sperm. The addition of lactoferrin was significantly (p<.05) effective on increasing values of progressive motility, straightness, and linearity in caput epididymal sperm and values of motility in cauda epididymal sperm. When ejaculated sperm were incubated in capacitation medium, percentages of motile and progressively motile sperm decreased largely within the first period of 30min, followed by only minor changes. However, the addition of lactoferrin significantly lessened the early decreases of these parameters and additionally promoted capacitation-dependent changes of chlortetracycline staining patterns (from F pattern to B pattern). In other experiments, when ejaculated sperm were exposed to oxidative stress with 100-µM H2 O2 , the addition of lactoferrin partially protected them from dysfunction of flagellar movement and loss of progressive movement. In final experiments with frozen-thawed samples incubated in the capacitation medium, the addition of lactoferrin effectively survived dying sperm and suppressed occurrence of sperm agglutination. These results may suggest biological and biotechnological potentials of lactoferrin for modulation of bovine sperm viability, motility, capacitation state, and preservation in vitro.