Kojic acid inhibits pig sperm apoptosis and improves capacitated sperm state during liquid preservation at 17°C.

The parameters of sperm apoptosis and capacitation during liquid storage at 17°C can indicate the quality of pig sperm and the potential development of early embryos. However, the effect of kojic acid (KA) on semen preservation and its mechanism has not been fully understood. In this study, we discovered that adding KA to the diluent improved the antioxidant capacity of sperm mitochondria, maintained the normal structure of sperm mitochondria, and reduced sperm apoptosis. Western blot analysis revealed that KA prevented the release of Cytochrome c from mitochondria to the cytoplasm, reduced the expression of pro-apoptosis proteins cleaved Caspase-3 and cleaved Caspase-9, and increased the expression of the antiapoptosis protein Bcl-XL. Furthermore, KA also enhanced the motility parameters, oxidative phosphorylation level, adenosine triphosphate level, and protein tyrosine phosphorylation of capacitated sperm, while preserving the acrosome integrity and plasma membrane integrity of capacitated sperm. In conclusion, this study offers new insights into the molecular mechanism of how KA inhibits porcine sperm apoptosis and improves capacitated sperm parameters. Additionally, it suggests that KA can serve as an alternative to antibiotics.

A new computational approach, based on images trajectories, to identify the subjacent heterogeneity of sperm to the effects of ketanserin.

The identification of kinematic subpopulations is of paramount importance to understanding the biological nature of the sperm heterogeneity. Nowadays, the data of motility parameters obtained by a computer-assisted sperm analysis (CASA) system has been used as input to distinct algorithms to identify kinematic subpopulations. In contrast, the images of the trajectories were depicted only as examples of the patterns of motility in each subpopulation. Here, python code was written to reconstruct the images of trajectories, from their coordinates, then the images of trajectories were used as input to a machine learning clustering algorithm of classification, and the subpopulations were described statistically by the motility parameters. Finally, the images of trajectories in each subpopulation were displayed in a way we called Pollock plots. Semen samples of boar sperm were treated with distinct concentrations of ketanserin (an antagonist of the 5-HT2 receptor of serotonin) and untreated samples were used as a control. The motility of sperm in each sample was analyzed at 0 and 30 min of incubation. Six subpopulations were found. The subpopulation 2 presented the highest values of velocities at 0 or 30 min. After 30 min of incubation, the ketanserin increased the values of the curvilinear velocity at high concentrations, whereas the linearity and the straight velocity decreased. Our computational model permits better identification of the kinematic subpopulations than the traditional approach and provides insights onto the heterogeneity of the response to ketanserin; thus, it could significantly impact the research on the relationship between sperm heterogeneity-fertility.

Sperm capacitation as a predictor of boar fertility.

Prediction of a boar's fertility level has great economic importance for sow herds. After standard sperm morphology and motility metrics are met, approximately 25% of boars have less than 80% conception rates. Due in part to the many factors involved in the fertilization process, a multifactorial model incorporating multiple relevant sperm physiology factors will likely lead to increased understanding of boar fertility. Here we review the current literature on boar sperm capacitation as a predictor of boar fertility. While limited, several studies have provided correlations between the percentage of sperm in an ejaculate that are capable of undergoing sperm capacitation in a chemically defined media and artificial insemination field fertility as well as proteome and other methods. Work summarized here underscores the need for further understanding of boar fertility.

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

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

Linseed oil in boar's diet improved in vivo fertility and antioxidant status.

The reactive oxygen species (ROS) which are produced during storage of boar semen are causing oxidative stress and leads to poor fertility. Also, tropical and sub-tropical weather condition adversely impacts the physicomorphological quality and fertility of boar sperm. The aim of this study was to examine the effects of feeding linseed oil to boar on its seminal attributes, sperm kinetics, biomarkers of antioxidant, fatty acid profile of seminal plasma (SP) and sperm and in vivo fertility. Six Hampshire crossbreed boars were fed with 90 ml linseed oil (LIN) whereas six Hampshire crossbreed boars were fed 90 ml canola oil (CON) for 16 weeks. Sperm quality was evaluated (60 ejaculates for each group; a total of 120 ejaculates) for motility, livability, abnormal morphology, acrosomal membrane integrity, hypo-osmotic swelling test (HOST) and sperm kinetic parameters by computer assisted semen analysis (CASA) at 0 h and at 72 h of storage at 17°C. Biomarkers of antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC) and malondialdehyde (MDA) were measured in SP and serum. Gas chromatography-mass spectrometry (GC-MS) was used for the estimation of fatty acid composition of SP and sperm. Boars fed with linseed oil had higher semen volume (p < .01) and more total sperm numbers (p < .01). Feeding linseed oil to boar enhanced seminal attributes (p < .05) at 0 h as well as at 72 h of storage. Linseed oil feeding (p < .01) improved biomarkers of antioxidants and significantly (p < .01) lowered the lipid peroxidation in serum and SP. Linseed oil feeding (p < .05) increased the proportion of alpha linolenic (ALA), arachidonic and docosahexaenoic (DHA) fatty acids in SP. The ratio of n-6 to n-3 fatty acids in sperm increased significantly (p < .01) in treatment group. Farrowing rate was significantly (p < .05) higher in treatment group. In conclusion, feeding linseed oil to boar improved the in vivo fertility, enhanced antioxidant capacity and increased the DHA content of SP and sperm.

Genetic group and swim-up processing affect SYBR Green real-time PCR-based porcine semen sex ratio in Landrace and its crossbred boars.

Determination of factors affecting sex ratio is important while considering application of sex ratio enrichment approach. Present study aimed to design a SYBR Green qPCR-based method for measurement of primary sex ratio and to evaluate different factors (genetic group, sire, spermiogenic cycle and processing layer) affecting boar sperm sex ratio. The qPCR was based on relative copy number analysis of sex chromosome-specific single copy gene fragments with an autosomal gene as reference and was evaluated using DNA dilution series from pigs with numerically normal karyotype. The sex ratio was estimated from genomic DNA samples isolated from boar semen collected from different genetic groups at different time points and different processing layers. The X chromosome frequencies of semen samples revealed significant effect of genetic group. However, significant variation was observed neither within same genetic group nor between ejaculates of different spermatogenic cycles. Among the processing techniques studied, swim-up technique produced a significant X sperm enrichment in comparison to control whereas, Percoll density gradient failed to show any significant difference among layers. The lower layer in swim-up technique was found to contain higher proportion of X sperms. The designed qPCR is found to be an easy, less time-consuming method and does not require high end laboratory facilities or the specialized expertise. The lower layer of swim-up processing has a scope for X sperm enrichment in boar semen with proper validation.

Odorant Receptor OR2C1 Is an Essential Modulator of Boar Sperm Capacitation by Binding with Heparin.

Heparin, a class of glycosaminoglycans (GAGs), is widely used to induce sperm capacitation and fertilization. How heparin induces sperm capacitation remains unclear. Olfactory receptors (ORs) which are G protein-coupled receptors, have been proposed to be involved in sperm capacitation. However, the interaction between ORs and odor molecules and the molecular mechanism of ORs mediating sperm capacitation are still unclear. The present study aimed to explore the underlying interaction and mechanism between heparin and ORs in carrying out the boar sperm capacitation. The results showed that olfactory receptor 2C1 (OR2C1) is a compulsory unit which regulates the sperm capacitation by recognizing and binding with heparin, as determined by Dual-Glo Luciferase Assay and molecular docking. In addition, molecular dynamics (MD) simulation indicated that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. Furthermore, we demonstrated that knocking down OR2C1 significantly inhibits sperm capacitation. In conclusion, we highlighted a novel olfactory receptor, OR2C1, in boar sperm and disclosed the potential binding of heparin to Pro287, a conserved residue in the transmembrane helices region 7 (TMH7). Our findings will benefit the further understanding of ORs involved in sperm capacitation and fertilization.

Pharmacological Evidence Suggests That Slo3 Channel Is the Principal K+ Channel in Boar Spermatozoa.

Sperm ion channels are associated with the quality and type of flagellar movement, and their differential regulation is crucial for sperm function during specific phases. The principal potassium ion channel is responsible for the majority of K+ ion flux, resulting in membrane hyperpolarization, and is essential for sperm capacitation-related signaling pathways. The molecular identity of the principal K+ channel varies greatly between different species, and there is a lack of information about boar K+ channels. We aimed to determine the channel identity of boar sperm contributing to the primary K+ current using pharmacological dissection. A series of Slo1 and Slo3 channel modulators were used for treatment. Sperm motility and related kinematic parameters were monitored using a computer-assisted sperm analysis system under non-capacitated conditions. Time-lapse flow cytometry with fluorochromes was used to measure changes in different intracellular ionic concentrations, and conventional flow cytometry was used to determine the acrosome reaction. Membrane depolarization, reduction in acrosome reaction, and motility parameters were observed upon the inhibition of the Slo3 channel, suggesting that the Slo3 gene encodes the main K+ channel in boar spermatozoa. The Slo3 channel was localized on the sperm flagellum, and the inhibition of Slo3 did not reduce sperm viability. These results may aid potential animal-model-based extrapolations and help to ameliorate motility and related parameters, leading to improved assisted reproductive methods in industrial livestock production.

Changes in aquaporins mRNA expression and liquid storage at 17°C: A potential biomarker of boar sperm quality?

Artificial insemination (AI) for pigs relies on liquid storage of extended semen at 17°C, which preserves sperm quality and ensures its fertilizing capacity. Routine quality controls include the evaluation of sperm motility, viability and capacitation status. The physiological functions of all these features depend on transmembrane aquaporins (AQPs), proteins playing key roles in osmoadaptation. In this study, we made a relative quantification, using RT-qPCR, of the mRNA of several sperm AQPs in AI-liquid semen doses before and after a 48-hr incubation period, aiming to determine possible quantitative compromising expression changes during the process that could serve as a diagnostic tool. Our results showed a decrease in classical sperm motility variables (total and progressive motility and velocity) and sperm viability after 48-hr storage, whereas capacitation status increased overtime. mRNA expression increased in the orthodox AQP4 and AQP6 after 48-hr incubation, relative to control (0 hr) and 24-hr time-points. Moreover, mRNA expression of aquaglyceroporins AQP3, AQP7 and AQP10 was higher after 48-hr incubation, confirmed by AQP7-protein validation using Western blot. Our results indicate that expression levels of AQPs-mRNA can change in ejaculated pig spermatozoa under conditions of ex-vivo incubation that could modify sperm homeostasis, suggesting it could eventually become a relevant molecular biomarker to assess the efficiency of liquid storage of pig semen.

Transcriptome-wide m6A profiling reveals mRNA post-transcriptional modification of boar sperm during cryopreservation.

BACKGROUND: Cryopreservation induces transcriptomic and epigenetic modifications that strongly impairs sperm quality and function, and thus decrease reproductive performance. N6-methyladenosine (m6A) RNA methylation varies in response to stress and has been implicated in multiple important biological processes, including post-transcriptional fate of mRNA, metabolism, and apoptosis. This study aimed to explore whether cryopreservation induces m6A modification of mRNAs associated with sperm energy metabolism, cryoinjuries, and freezability. RESULTS: The mRNA and protein expression of m6A modification enzymes were significantly dysregulated in sperm after cryopreservation. Furthermore, m6A peaks were mainly enriched in coding regions and near stop codons with classical RRACH motifs. The mRNAs containing highly methylated m6A peaks (fts vs. fs) were significantly associated with metabolism and gene expression, while the genes with less methylated m6A peaks were primarily involved in processes regulating RNA metabolism and transcription. Furthermore, the joint analysis of DMMGs and differentially expressed genes indicated that both of these play a vital role in sperm energy metabolism and apoptosis. CONCLUSIONS: Our study is the first to reveal the dynamic m6A modification of mRNAs in boar sperm during cryopreservation. These epigenetic modifications may affect mRNA expression and are closely related to sperm motility, apoptosis, and metabolism, which will provide novel insights into understanding of the cryoinjuries or freezability of boar sperm during cryopreservation.

Transcriptome analysis identifies genes and co-expression networks underlying heat tolerance in pigs.

BACKGROUND: Heat stress adversely affects pig growth and reproduction performance by reducing feed intake, weight gain, farrowing rate, and litter size. Heat tolerance is an important characteristic in pigs, allowing them to mitigate the negative effects of heat stress on their physiological activities. Yet, genetic variation and signaling pathways associated with the biological processes of heat-tolerant pigs are currently not fully understood. This study examined differentially expressed genes and constructed gene co-expression networks on mRNAs of pigs under different heat-stress conditions using whole transcriptomic RNA-seq analyses. Semen parameters, including total sperm number per ejaculate, motility, normal morphology rate, droplets, and rejected ejaculate rate, were measured weekly on 12 boars for two time periods: thermoneutral (January to May), and heat stress (July to October). Boars were classified into heat-tolerant (n = 6) and heat-susceptible (n = 6) groups based on the variation of their ejaculate parameters across the two periods. RNA was isolated from the blood samples collected from the thermoneutral and heat stress periods for gene expression analysis. RESULTS: Under heat stress, a total of 66 differentially expressed genes (25 down-regulated, 41 up-regulated) were identified in heat-tolerant pigs compared to themselves during the thermoneutral period. A total of 1041 differentially expressed genes (282 down-regulated, 759 up-regulated) were identified in the comparison between heat-tolerant pigs and heat-susceptible pigs under heat stress. Weighted gene co-expression network analysis detected 4 and 7 modules with genes highly associated (r > 0.50, p < 0.05) with semen quality parameters in heat-tolerant and heat-susceptible pigs under the effects of heat stress, respectively. CONCLUSION: This study utilized the sensitivity of semen to heat stress to discriminate the heat-tolerance ability of pigs. The gene expression profiles under the thermoneutral and heat stress conditions were documented in heat-tolerant and heat-susceptible boars. Findings contribute to the understanding of genes and biological mechanisms related to heat stress response in pigs and provide potential biomarkers for future investigations on the reproductive performance of pigs.

Rosmarinic acid improves boar sperm quality, antioxidant capacity and energy metabolism at 17°C via AMPK activation.

Boar sperm are susceptible to oxidative damage caused by reactive oxygen species (ROS) during storage. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important therapeutic target, because it is a cellular metabolism energy sensor and key signalling kinase in spermatozoa. We evaluated the effects of rosmarinic acid (RA), an antioxidant, on boar sperm during liquid storage to determine whether it protects boar sperm via AMPK activation. Boar ejaculates were diluted with Modena extender with different concentrations of RA and stored at 17°C for 9 days. Sperm quality parameters, antioxidant capacity, energy metabolism, AMPK phosphorylation and fertility were analysed. Compared with the control, 40 µmol/L significantly improved sperm motility, plasma membrane integrity and acrosome integrity (p < .05). The effective storage time of boar sperm was up to 9 days. On the third and seventh days, the sperm with RA exhibited increased total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, adenosine triphosphate (ATP) content, mitochondrial membrane potential (ΔΨm) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, whereas malondialdehyde (MDA) content was significantly decreased (p < .05). Western blot showed that RA, as well as AICAR (AMPK activator), promoted AMPK phosphorylation, whereas Compound C (AMPK inhibitor) inhibited this effect. The sperm-zona pellucida binding experiment showed that 40 µmol/L RA increased the number of sperm attached to the zona pellucida (p < .05). These findings suggest meaningful methods for improved preservation of boar sperm in vitro and provide new insights into the mechanism by which RA protects sperm cells from oxidative damage via AMPK activation.

HVCN1 Channels Are Relevant for the Maintenance of Sperm Motility During In Vitro Capacitation of Pig Spermatozoa.

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca2+ entrance to the sperm head.

Boar sperm quality and oxidative status as affected by rosmarinic acid at 17 °C.

Peroxidation damage induces sublethal injury to boar sperm during preservation. Rosmarinic acid (RA) has already been verified to efficiently protect cells from oxidant-induced injury and to produce significant effect on cryopreservation of semen. Through our experiments, we aim at investigating whether RA has a positive effect on the preservation of pig semen at room temperature. The semen collected from sexually mature Large White boars were preserved at 17 °C in Beltsville thawing solution (BTS) supplied. The boar sperm were exposed to 0, 25, 50, 75, 100, 125 and 150 µM RA in vitro and the sperm functions were examined. The sperm motility, the acrosome and plasma membrane integrity, the catalase activity (CAT), the total antioxidative capacity (T-AOC) activity and the malondialdehyde content (MDA) were examined at 0, 1, 3 and 5 days. The BTS diluent containing RA improved the sperm quality during the process of liquid preservation compared with the control treatment. After 5 days of liquid preservation, the addition of RA at 100 µM produced an optimal effect on the survival time as well as on the maintenance of motility, acrosome and plasma membrane integrity; T-AOC activity; CAT activity; and the MDA content. Besides, our results in the reproductive experiments showed that the addition of RA at 100 µM to the BTS diluent increased the pregnancy rate. These results suggest that the proper concentration of RA in boar semen extenders possibly improves the artificial insemination efficiency by reducing the sperm damage and the subsequent dysfunction during liquid preservation in swine production systems.

"In vitro" capacitation and further progesterone-induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa.

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone-induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono- and bi-dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono-dimensional Western blot in non-capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi-dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non-capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.

Exploration of miRNA and mRNA Profiles in Fresh and Frozen-Thawed Boar Sperm by Transcriptome and Small RNA Sequencing.

Due to lower farrowing rate and reduced litter size with frozen-thawed semen, over 90% of artificial insemination (AI) is conducted using liquid stored boar semen. Although substantial progress has been made towards optimizing the cryopreservation protocols for boar sperm, the influencing factors and underlying mechanisms related to cryoinjury and freeze tolerance of boar sperm remain largely unknown. In this study, we report the differential expression of mRNAs and miRNAs between fresh and frozen-thawed boar sperm using high-throughput RNA sequencing. Our results showed that 567 mRNAs and 135 miRNAs were differentially expressed (DE) in fresh and frozen-thawed boar sperm. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the majority of DE mRNAs were enriched in environmental information processing such as cytokine-cytokine receptor interactions, PI3K-Akt signaling, cell adhesion, MAPK, and calcium signaling pathways. Moreover, the targets of DE miRNAs were enriched in significant GO terms such as cell process, protein binding, and response to stimuli. In conclusion, we speculate that DE mRNAs and miRNAs are heavily involved in boar sperm response to environment stimuli, apoptosis, and metabolic activities. The differences in expression also reflect the various structural and functional changes in sperm during cryopreservation.

A mechanism by which Astragalus polysaccharide protects against ROS toxicity through inhibiting the protein dephosphorylation of boar sperm preserved at 4 °C.

Numerous studies have shown that Astragalus polysaccharide (APS) has strong antioxidant effects and high practical value for preserving semen at low temperatures in vitro. However, to date, little attention has been paid to the precise mechanism of APS in sperm preservation at 4 °C. Thus, to gain further insight into the protective effects of APS, the present study was performed to assess the changes in sperm quality parameters, antioxidant capacity, ATP content, and protein phosphorylation levels. Here, we demonstrated that supplementation with APS could effectively preserve boar sperm quality parameters such as sperm motility, acrosome integrity, and mitochondrial membrane potential. Moreover, we found that the positive effects of APS on boar sperm quality were mainly due to the elimination of excessive mitochondrial ROS, the improvement of antioxidant capacities and the enhancement of ATP levels. Interestingly, by conducting a series of studies on protein phosphorylation, we also discovered that APS could protect boar sperm from oxidative stress and energy deficiency through inhibiting the protein dephosphorylation caused by ROS via the cAMP-PKA signaling pathway. To our knowledge, this is the first exploration of the molecular mechanism underlying the protective roles of APS toward ROS toxicity from the perspective of energy metabolism and protein modification. This study comprehensively provides novel insights into the action mechanism of the protective effects of antioxidants on sperm stored at 4 °C and reveals the practical feasibility of using APS as a boar semen extender supplement for assisted reproductive technology.

Boar sperm quality after supplementation of diets with omega-3 polyunsaturated fatty acids extracted from microalgae.

This study evaluated effects of diet supplementation with omega-3 polyunsaturated fatty acids (PUFA) from microalgae on boar sperm quality. Two groups of boars (n = 3 each) were fed during 75 days either a commercial diet (control), or the same diet supplemented with omega-3 PUFA from the heterotrophic microalgae Schizochytrium sp. (120 g/kg). Sixteen ejaculates were collected per boar. Some sperm kinetics parameters were inferior for supplemented than for control boars (p < .05): distance average path; distance in both curved and straight line; velocity average path, velocity in both curved and straight line; and amplitude of lateral head displacement. Spermatozoa from supplemented boars presented lower mitochondrial functionality, but greater membrane fluidity compared to the control group (p < .01). Membrane and acrosome integrity, production of reactive oxygen species and lipid peroxidation did not differ (p > .05). Serum cholesterol levels were greater (p < .05) for supplemented than for control boars at the 30th and 60th d of supplementation, but levels of triglycerides and IGF-1 did not differ (p > .05). Compared to the control, spermatozoa of supplemented boars were slower, travelled shorter distances and presented impaired energy metabolism, but their greater membrane fluidity may potentially favour their cryopreservation.

Effect of boar ejaculate fraction, extender type and time of storage on quality of spermatozoa.

The aim of this study was to investigate the effect the sperm-rich fraction (F1) and the post-F1 fraction (F2) on the quality of boar spermatozoa stored in a liquid state. Ejaculates were collected from three Polish Landrace boars. Each ejaculate fraction was diluted with BTS short-term extender and Safe-Cell Plus (SCP) long-term extender and stored for seven days (D1-D7) at 17°C. Analyses included sperm motility parameters, normal apical ridge (NAR) acrosomes and plasma membrane integrity (PMI). Prior to the dilution of fractions, marked changes (p<0.05) were noted between F1 and F2 in progressive motility (PMOT), velocity average pathway (VAP) and velocity straight line (VCL). After the ejaculate was diluted, the type of fraction and type of extender significantly affected (p<0.05) PMOT, being markedly higher (p<0.05) for F1 extended in BTS. No marked changes (p<0.05) were observed between F1 and F2 extended in SCP for any of the analyzed sperm quality parameters during seven days of storage. Significantly higher (p<0.05) values of sperm quality parameters were noted in F1 compared with F2 for BTS on D7 of storage. The results of the four-way ANOVA analysis indicate that boar, fraction of ejaculate, extender type and day of storage had significant effects on the quality of boar stored spermatozoa. The F1 was characterised by higher quality of spermatozoa during storage in comparison with F2 in the short-term extender. Using the long-term extender containing the proteins allowed for a better application of F2, which could be important for the pig industry.

Selected steps of swine ICSI method to overcome seasonal effect and achieve acceptable early embryonic development, a preliminary study.

Porcine intra cytoplasmic sperm injection's (ICSI) efficacy by selected protocol steps was investigated. Three trials per year's period (hot, medium, cold) were carried out. Only large size follicles (6-8mm) were aspirated, brilliant cresyl blue (BCB) test was performed and only the BCB+ oocytes were in vitro maturated (40h) and involved to ICSI process. The presumptive embryos were in vitro cultured (15h). Raw boar semen and SpermCatch® as slowing medium were used. No differences were observed between periods regarding early embryonic development and maturation competence. ICSI achieves acceptable porcine early embryonic development rates under the investigated conditions.