Pig Semen Research Articles

Thyroxine regulates pig Sertoli cell line proliferation and maturation through the IKK/NFκB and p38 MAPK signaling pathways

The aim of this study was to investigate the signaling pathways involved in the proliferation and differentiation of pig Sertoli cells (SCs) mediated by thyroid hormone (T3) to provide a theoretical and practical basis for enhancing pig semen production. The effects of different concentrations of T3 on the proliferation of pig SCs were evaluated using the CCK8 assay. The impact of T3 on the proliferation and differentiation of pig SCs was further examined using RNA-seq, qPCR, and Western Blotting techniques. Additionally, the involvement of the p38 MAPK and NFκB pathways in mediating the effects of T3 on SCs proliferation and differentiation was investigated. Our findings revealed a strong correlation between the dosage of T3 and the inhibition of pig SCs proliferation and promotion of maturation. T3 regulated the activation state of the NFκB signaling pathway by upregulating IKKα, downregulating IKKβ, and promoting IκB phosphorylation. Furthermore, T3 facilitated SCs maturation by upregulating AR and FSHR expression while downregulating KRT-18. In conclusion, T3 inhibits pig SCs proliferation and promote pig SCs maturation through the IKK/NFκB and p38 MAPK pathways. These findings provide valuable insights into the mechanisms by which T3 influences the proliferation and maturation of pig SCs.

Evaluasi Kerusakan DNA Spermatozoa Semen Cair Babi Menggunakan Pewarnaan Toluidine Blue

The integrity of the deoxyribonucleic acid (DNA) in sperm plays a crucial role in the fertilization process and embryonic development. This study aimed to evaluate the level of the DNA fragmentation of boar sperm in tris-citrate-fructose extender preserved at temperatures between 16-18°C for 3 days. The level of sperm DNA damage was analysed using toluidine blue (TB) staining. Pig semen collected from 4 male pigs aged 1.3-1.8 years was used as the research sample. Semen collected was conducted twice a week. The experimental method using a Completely Randomized Design with 4 storage duration groups as follows: Group I (0 hours of storage), Group II (24 hours of storage), Group III (48 hours of storage), and Group IV (72 hours of storage), each with 5 replications. The data were analysed using analysis of variance (ANOVA) and, if significant differences were found, Duncan's test was performed. The results of the evaluation of the percentage of DNA damage during the storage process at 16-180C at 0 hours, 24 hours, 48 hours and 72 hours was: 0.33 ± 1.66%, 0.80 ± 0.447%, 1.60 ± 1.14% and 2.80 ± 0.447%, respectively. These results demonstrate that liquid boar semen preserved with tris citrate fructose extender at 16-18 °C for 3 days has spermatozoa DNA damage levels within the normal range, making it suitable for artificial insemination and ensuring optimal AI success.

Respons Kualitas Semen Babi Terhadap Penambahan PGF2α

This study aimed to determine the response of pig semen quality to the addition of PGF2 during semen dilution. The research was conducted from August to December 2022 at one of the pig farms in Tomohon City, North Sulawesi. The study was conducted using 4 (four) variations of PGF2 doses namely 0, 20, 40, 60 μg/mL and 5 (five) repetitions. The results obtained were then tested with the One Way Anova test and if the Sig value. <0.05 then it will be continued with Duncan's Multiple Range Test. The data processed is primary data obtained during the research. There are four treatments and five repetitions conducted by the author when conducting this research. The results of the one way anova test showed that there was no effect of treatment on mass motion and conception of pig semen. The results of the one way anova test on motility showed the effect of treatment at the +60µg/100ml prostaglandin treatment level. The results of the Duncan test on the three parameters did not show a real effect of each treatment on the parameters tested. The conclusion of the addition of PGF2α as much as 20µg/mL, 40µg/mL, PGF2α as much as 20µg/mL, 40µg/mL, 60µg/mL in pig semen (Sus Scrofa) has no effect on mass movement in pig semen (Sus Scrofa) has no effect on mass movement and conception of pigs but the addition of 60µg/mL in pig semen (Sus Scrofa) has a significant effect on individual sperm movement of pigs.
 

Evaluation of Motility, Viability and Abnormality of Boar Spermatozoa in Various Modified Extenders

The purpose of this study was to conduct an assessment on the quality of pig semen spermatozoa during storage and determine which diluent material should be used and how long pig semen can be stored. Freshly ejaculated boar semen was obtained from a two-years-old Duroc male and was diluted with Beltsville Thawing Solution (BTS) and Tris Citrate Fructose (TCF) supplemented with 5% egg yolk (KT) and extract of Borassus flabellifer Linn mesocarp (EM) 0.01% and stored at 13°C for up to 4 days. An experimental study with a completely randomized design with four groups and five repeats was applied. Parameters observed were motility, viability and abnormality of spermatozoa. Sperm quality was evaluated by examining motility and abnormality using a phase-contrast microscope with a magnification of 100X, and viability by eosin nigrosine staining. Parametric data were analyzed with ANOVA followed by Duncan test. The results showed that the supplementation of 5% egg yolk in the BTS diluent-based treatment group showed a higher percentage of motility (P<0.05) than without egg yolk, while the TCF-based diluent group showed no differences (P<0.05). Modified-Tris Citrate Fructose diluent supplemented with 5% egg yolk and 0.01% mesocarp extract was able to maintain a significantly higher percentage of viability (67.50±2.0) (P<0.05) than other groups, and had a percentage of abnormalities (9.75±0.957), significantly lower (P<0.05) than other groups. It was concluded that the modification of diluent TCF with 5% egg yolk and mesocarp extract 0.01% provide the best results in maintaining sperm quality with the highest percentage of sperm motility and viability, and the lowest percentage of sperm abnormality compared to the other three diluents. Likewise, all groups of modified-BTS and TCF diluent supplemented with 5% egg yolk and extract of Borassus flabellifer Linn mesocarp in this study were able to maintain motility >40%, viability >45% and abnormalities <20% so they were appropriate for using in the AI procedure.

Addition of ellagic acid improved the immune ability and delayed the apoptosis of ovarian granulosa cells of Guizhou black goat

Context Follicular development plays an important role in the growth and reproduction of female mammals. Ellagic acid (EA), as a natural antioxidant, has been used in freezing protection of pig semen. However, the effects of EA on immunity and the anti-apoptotic ability of ovarian granulosa cells (GCs) are still unclear. Aims The aim of this study was to analyse the effects of different concentrations of EA on the immune and anti-apoptotic ability of ovarian GCs of Guizhou black goats. Methods In this study, different concentrations of EA (0, 50, 100, 150, 200 μmol/L) were added to the culture of ovarian GCs in vitro, and Cell-Counting Kit 8 (CCK8) assay, cell wound scratch assay, and real-time fluorescence quantitative polymerase chain reaction (RT–qPCR) assay were used to detect the effects of different concentrations of EA on the proliferation, migration, and reproductive marker genes of ovarian GCs. Then the optimal addition concentration of EA was selected and the effects of EA supplementation on immune factors, cytochrome P450 family 19 subfamily A member 1 gene (CYP19A1), estradiol concentrations, intracellular reactive oxygen species concentrations, and apoptosis-related protein expression were detected by RT–qPCR, enzyme-linked immunosorbent assay (ELISA), ROS, and western blotting on the basis of the optimal addition concentration. Key results The CCK8 test and cell scratch test showed that the addition of EA could significantly inhibit the proliferation and migration ability of ovarian GCs compared with the control group, and a dose effect was observed with the increase in concentration. RT–qPCR results showed that different concentrations of EA significantly increased the expression of genes associated with reproduction, including bone morphogenetic protein 15 (BMP15), bone morphogenetic protein receptor 1B (BMPR-1B), growth differentiation fFactor 9 (GDF9), and follicle-stimulating hormone β subunit (FSHβ), and the maximum increase was observed at 150 μmol/L EA. Further analyses using 150 μmol/L EA as the optimal concentration showed significantly increased expressions of CYP19A1, interleukin-10 (IL-10), and superoxide dismutase (SOD2) after EA supplementation, while the expression of IL-8 was significantly decreased compared with those of the control group. ELISA and ROS showed that both intracellular and extracellular estradiol concentrations were higher, while ROS concentrations were significantly lower than those in the control group. Western blotting results showed that 150 μmol/L EA significantly decreased the expression of Caspase-3 and Caspase-9 and the ratio of BCL2-associated X:B-cell lymphoma-2. Conclusions The supplementation of 150 μmol/L EA had significant effects on improving GC immunity and delaying GC apoptosis in goats. The addition of EA also increased the expression of BMP15, BMPR-1B, GDF9, FSHβ, and CYP19A1 and promoted the secretion of estradiol in GCs. Implications These results provided a preliminary lead for further research on the effect of EA on the maturation and development of goat oocytes in vitro.

Inhibition of HSP70 during prolonged liquid storage at 17 °C increases mitochondrial membrane potential and calcium levels

Serine phosphorylation of heat shock protein 70 (HSP70/HSPA1A) is known to be involved in sperm cryotolerance. This study aimed to determine whether HSP70 is implicated in the resilience of pig semen to prolonged storage at 17 °C. For this purpose, HSP70 was blocked with various concentrations of YM-1 (0, 0.05, 0.1 and 0.2 µM), an allosteric inhibitor of this chaperone, and samples were stored for 21 d. At various time points (0, 4, 10, 14, and 21 d), sperm motility and kinetic parameters by computer-assisted semen analysis, and viability (SYBR14/PI), mitochondrial membrane potential (JC1) and intracellular levels of calcium (FLUO3), total ROS (H2DCFDA) and superoxides (HE) through flow cytometry were assessed. No significant differences in sperm motility parameters were observed, except a reduction of VSL in the treatment containing 0.2 µM YM-1 at day 14 (P<0.05). Although no YM-1 concentration affected viability and intracellular concentrations of total ROS and superoxides, inhibiting HSP70 with YM-1, regardless of inhibitor concentration, increased intracellular calcium concentrations at day 10 compared to the control (P<0.05). Moreover, the treatment containing 0.05 µM YM-1 had higher JC1agg/JC1mon ratios than the control at day 10 (P<0.05). In conclusion, although inhibition of HSP70 with YM-1 did not affect pig sperm survival during long-term storage at 17 °C, it increased mitochondrial activity and intracellular calcium concentrations. This work was supported by the Ministry of Science and Innovation, Spain (PID2020-113320RB-I00) and the Regional Government of Catalonia, Spain (2017-SGR-1229).

The effects of red LED light on pig sperm function rely upon mitochondrial electron chain activity rather than on a PKC-mediated mechanism.

While irradiation with red LED light has been reported to modulate sperm function in different mammalian species, the mechanisms underlying their response are poorly understood. This work sought to provide new insights into whether this effect relies on a direct action upon mitochondrial electron chain and/or on PKC-linked mechanisms such as those related to opsins. For this purpose, pig semen was light-stimulated for 1, 5 or 10 min in the presence/absence of antimycin A, an inhibitor of the mitochondrial electron chain, or PKC 20–28® (PKCi), a PKC inhibitor. Antimycin A completely blocked the effects of light at all the performed irradiation patterns. This effect was linked to a complete immobility of sperm, which was accompanied with a significant (p < 0.05) drop in several markers of mitochondrial activity, such as JC-1 staining and O2 consumption rate. Antimycin A, however, did not affect intracellular ATP levels, intramitochondrial calcium, total ROS, superoxides or cytochrome C oxidase (CCO) activity. In the case of PKCi, it did also counteract the effects of light on motility, O2 consumption rate and CCO activity, but not to the same extent than that observed for antimycin A. Finally, the effects observed when sperm were co-incubated with antimycin A and PKCi were similar to those observed with antimycin A alone. In conclusion, red LED light acts on sperm function via a direct effect on mitochondrial electron chain. Additionally, light-activated PKC pathways have a supplementary effect to that observed in the electron chain, thereby modulating sperm parameters such as motility and CCO activity.

PENGARUH PERBEDAAN WAKTU EKUILIBRASI TERHADAP KUALITAS SEMEN BEKU BABI LANDRACE DALAM PENGENCER DURASPERM MODIFIKASI DENGAN AIR BUAH LONTAR (Effect of different equilibration time on the quality of landrace boar frozen semen on modification diluent....

An experiment was carried out to recognize the effect of long equilibration time on the quality of spermatozoa and to obtain optimal equilibration time in the process cryopreservation of landrace pig semen. Semen was obtained from a landrace boar aged 2-3 years using glove hand method. The semen was than evaluated macroscopically and microscopically, good quality semen was diluted in a basic diluent, than kept for two hours at a temperature of 27-28oC. The semen were then centrifuged at 2000 rpm for 15 minutes, the supernatant was discarced and the pellets was re-diluted into basic diluent with a ratio of 1:1. Then, semen was diluted in treatment diluent with modified durasperm + 6% lactose + 4% glycerol with a concentration of 500 x 106/0.5 mL. Furthermore, the semen was packaged in 0.5 ml straws, then equilibrated at a temperature of 3-5oC appropriate to treatment, with 1 hour equilibration (T1), 2 hours equilibration (T2) and 3 hours equilibration (T3). After equilibration, the straws was frozen horizontally 5 cm above the liquid N2 vapor for 10 minutes at a temperature of ± -110oC and then stored in a liquid N2 container (-196oC). To test frozen semen, it was done by stored frozen semen at 37oC for 30 seconds. The results showed that the highest quality of spermatozoa (P &lt;0.05) was found in T2 and T3 when compared to T1 (P&gt; 0.05), with the motility value was T2: 28.13 ± 2.39%; T3: 25.00 ± 4.08%; and T1 18,15±3,23%. It can be concluded that 2 hours and 3 hours equilibration time was able to maintain the quality of frozen semen landrace boars and the statistically was not significant.

Astaxanthin improves preserved semen quality at 17°C by enhancing sperm antioxidant and motility properties.

Porcine sperm is rich in polyunsaturated fatty acids; therefore, it is highly susceptible to oxidative damage during storage. Inhibition of oxidative stress during preservation is essential for maintaining sperm motility. Astaxanthin is a potent antioxidant used in the cosmetic and pharmaceutical industries. This study aimed to explore the effect of supplementing astaxanthin as an extender of porcine semen preservation dilutions at 17°C. Various concentrations of astaxanthin were added to diluted porcine semen at 17°C. We performed computer-assisted semen analysis, evaluation of plasma membrane integrity and acrosome integrity, and measurement of total antioxidant activity, malondialdehyde (MDA) content, reactive oxygen species levels, superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GSH-PX) activity and sperm motility parameters. Compared with the control group, the addition of 0.25 μg/ml astaxanthin group significantly improved sperm motility parameters stored on the fifth day; these were increased levels of sperm SOD, GSH-PX and CAT (p < .05), increased sperm adenosine trisphosphate and lactate dehydrogenase levels and decreased sperm MDA levels (p < .05). These findings suggest that adding 0.25 μg/ml of astaxanthin improves the quality of porcine semen stored at 17°C. Our findings provide theoretical support for developing new protective agents critical for preserving pig semen at 17°C.

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 (0hr) 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.

Effect of homeopathic treatment used in commercial boar semen diluent on sperm viability

Background: It has been speculated that the homeopathic treatment of sperm cells in order to improve semen quality could be promising. However, few data is available and its use in spermatozoa requires investigation. It is well established that mitochondrial membrane potential is an important viability parameter of spermatozoa and it is intimately related to reproductive efficiency. In this manner, new technologies in order to improve the activity of sperm cells and, finally, the fecundity of swine herds are of extremely importance. Due to the lack of knowledge of homeopathic treatment effect on spermatozoa, the aim of the present study was to verify the effect of three different homeopathic treatments on viability of boar sperm cells. &#x0D; Methods: semen samples were obtained from two sexually mature boars (18 mo of age). The boars were cross bred, with similar genetics of Pietrain versus Duroc, BP 450 progeny from a supplier company of similar reproductive performance animals. The animals were maintained in individual stalls, study conducted in Sao Paulo - Brazil. Three homeopathic treatments: Pulsatilla 6CH, Avena 6 CH or both, compared to placebo treatment (sucrose), the homeopathic medicaments or the control were administrated as globules manipulated according Brazilian Homeopathic Pharmacology. Each globule weighted 30 mg and contained sucrose as vehicle. One dose of two globules was added per 100 mL of diluted boar semen, which were chilled for 24 or 48 hours. All samples were labeled in codes in order to allow all laboratory analysis and evaluations being performed as a blind test. Data were tested for normality of residues and homogeneity of variances using the Guided Data Analysis software. Variables and interactions were analyzed by the PROC MIXED of the SAS package (SAS Institute Ins. Cary, NC). Adjusted least squares means (LSMEANS) of treatments were compared using the Tukey Test. &#x0D; Results: The different treatments contributed to maintain acrossome integrity for prolonged periods of cooling over 48 hours. The use of Pulsatilla was effective in maintaining high sperm mitochondria activity up to 24 hours from harvesting.&#x0D; Conclusion: Homeopathic medications can be used in artificial insemination in order to improve the quality of cooled and stored pig semen [1].&#x0D; Keywords: homeopathy, swine semen, sperm viability.&#x0D; Reference&#x0D; [1] Soto, F. R. M.; Vuaden, E. R.; Coelho, C. P.; Bonamin, L. V.; Azevedo, S. S. A.; Benites, N. R.; Barros, F. R. O.; Goissis, M. D.; Assumpção, M. E. O. D.; Visintin, J. A.; Marques, M. G. Effects of the utilization of homeopathic elements in commercial diluent on swine sperm viability. In Vitro Cell.Dev.Biol.—Animal. 47:205–209, 2011.

Effect of Antioxidants on Pig Semen Cryopreservation to Preserve Sperm Fertility after Thawing

Boar semen cryopreservation has a high potential in the swine industry, allowing the large-scale use of genetically superior animals, improving efficiency, product quality, helping to reduce the risk of disease spread and gathering needs from the market. From a genetic point of view, semen freezing is desirable for genetic diversification, favouring a more efficient reproduction as well as the constitution of germplasm banks, including for repopulation in case of disease outbreak. However, freezing this semen for long periods for practical use is limited by the reduced viability and fertilization potential caused to sperm during the cryopreservation process and consequently low conception rates and smaller litters after artificial insemination. In part, the decrease in the fertilizing power of frozen spermatozoa may be associated with oxidative damage due to excessive formation of Reactive Oxygen Species (ROS), osmotic stress and cell damage due to ice formation during cryopreservation. To suppress the damage caused by ROS, the present study was conducted to determine the impact of supplementation with three antioxidants, these being ascorbic acid, a-tocopherol and reduced glutathione, evaluating the parameters of semen quality, viability, total and progressive motility, vigour and agglutination rate after thawing. For this purpose, semen was collected from five boars, each being collected three times, at weekly intervals, always at the same time. Immediately after harvesting, the macroscopic (colour, appearance, and volume) and microscopic evaluation of the semen (mass motility, concentration, progressive individual motility, spermatic vigour and spermatic morphology) were evaluated. Subsequently, the semen was placed at 15°C for two hours and centrifuged at 800 x g for 10 minutes also at 15°C, removing the supernatant. For the freezing medium, a base medium consisting of a commercial MR-A extender, supplemented with 3% v/v glycerol, 10% v/v egg yolk and 0.20% w/v Sodium Dodecyl Sulfate (SDS) was used. The nine treatments used in the study were, respectively, ascorbic acid at concentrations of 100, 200 and 400μL, a-Tocopherol at concentrations of 200, 400 and 800μM and reduced Glutathione at concentrations of 2.5, 5 and 10 mg/l and numbered as T1 to T9, respectively. In the control group, semen was frozen in a medium without adding any antioxidant. The semen belonging to the different treatments and to the control was placed in 0.25ml insemination French straws and incubated at 6°C for two hours. The subsequent freezing was carried out in nitrogen vapours (-120°C) for ten minutes and immersed in liquid nitrogen after this period. After 7 days, the semen was thawed in a water bath at 37°C for 20 seconds, the straws dried on paper, placed on a microscope slide heated to 37°C and evaluated according to the parameters described above. Regarding the comparison between the different treatments, it was observed that the sperm viability obtained in the treatments with ascorbic acid as well as glutathione reduced, was not statistically different from the control group. Higher values of ascorbic acid and reduced glutathione reduced sperm viability after thawing. As for the use of a-tocopherol at a concentration of 400μM, the best results of the entire study were obtained, with sperm viability of 31.52% (±1.50). Regarding sperm motility and agglutination rate, a-tocopherol also showed the best results at the concentration of 200μM, in which the mean sperm motility was 2.57 ± 0.15 and 2.07 ± 0.15, respectively. The results of the present study allow us to infer that the addition of 200μM or 400μM of a-tocopherol to the swine semen-freezing medium has a positive effect on sperm viability parameters after thawing.

Pores Formation in Porcine Sperm Incubated in Streptolysin O and Its Post Thawing Viability after Trehalose Treatment

Background: Streptolysin O (SLO), a pore-forming protein in plasma membrane (PM), has been used to internalize a variety of molecules (DNA and RNA) in cells. In sperm, however, SLO has only been used to release acrosomal contents. Its possible use as biotechnology in the cryopreservation of pig semen. However, porcine sperm are very sensitive to the freeze-thaw process. The study aimed to evaluate the pore formation in the PM, the addition of trehalose and the post cryopreservation viability of porcine spermatozoa using SLO.Methods: Research period was spring 2017- summer 2018. Thirty ejaculates from five mature boars were used. Semen was incubated in SLO 0.6 IU/ml and trehalose (added at 100, 200 and 400 ìM). Semen diluted in commercial diluent as control group. Presence of pores was checked by scanning electronic microscopy. To evaluate sperm membrane integrity and functional status the Coomassie stain with HOST test and the Chlortetracycline test were used. Result: It was found that SLO could form pores in the sperm cell membrane The addiction of 200 ìM trehalose to the freezing medium have different effects on the quality of boar sperm, showing highest motility and viability during the cooling process.

Red LED Light Acts on the Mitochondrial Electron Chain of Mammalian Sperm via Light-Time Exposure-Dependent Mechanisms.

This work analyzes the effects of red LED light on mammalian sperm mitochondrial function, using the pig as an animal model. Liquid-stored pig semen was stimulated with red-light for 1, 5 and 10 min in the presence or absence of oligomycin A, a specific inhibitor of mitochondrial ATP synthase, or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific disruptor of mitochondrial electron chain. Whereas exposure for 1 and 5 min significantly (p < 0.05) decreased total motility and intracellular ATP levels, irradiation for 10 min induced the opposite effect. Oligomycin A abolished the light-effects on intracellular ATP levels, O2 consumption and mitochondrial membrane potential, whereas compared to non-irradiated samples, FCCP significantly (p < 0.05) increased O2 consumption when sperm were irradiated for 1 min. Both oligomycin A and FCCP significantly (p < 0.05) decreased total motility. Red-light increased cytochrome c oxidase activity with a maximal effect after 5 min of irradiation, which was abolished by both oligomycin A and FCCP. In conclusion, red-light modulates sperm mitochondrial function via electron chain activity in an exposition, time-dependent manner.

Identification of the major rabbit and guinea pig semen coagulum proteins and description of the diversity of the REST gene locus in the mammalian clade Glires.

The seminal vesicle secretions of guinea pig and rabbit were analyzed for semen coagulum proteins. Using SDS-PAGE we discovered a previously not fully recognized semen coagulum protein, Svp5, in the guinea pig and a single predominant component, SVP200, in the rabbit. Potential genes of these proteins were identified in genome databases by their homology with human and murine genes. The structure of their fullength transcripts was determined using seminal vesicle cDNA and sequencing primers based on genomic sequences. Homology searching indicated that both Svp5 and SVP200 were synthesized from composite genes that were the result of merger between two genes showing homology with human SEMG2 and PI3. For a deeper understanding of the evolution of the genes, we retrieved and analyzed genome sequences from the REST gene loci, encompassing genes of semen coagulum proteins and related rapidly evolving seminal vesicle-transcribed genes, of 14 rodents and 2 lagomorphs. The analysis showed that rodents of the suborders myomorpha, hystricomorpha, and castorimorpha had unique sets of REST genes, whereas sciuromorpha seemed to be lacking such genes. It also indicated a closer relationship between myomorpha and castorimorpha than to rodents of the two other analyzed suborders. In the lagomorph species, the pika appeared to be devoid of REST genes, whereas the rabbit had a single expressed REST gene, SVP200, and two pseudogenes. The structural similarity of semen coagulum proteins in rabbit and hystricomph species suggests that they are closely related. This was also supported by other similarities at their REST gene loci, e.g. the finding of a PI3-like gene in the rabbit that also had features in common with caltrin2 of hystricomorph rodents. The homologies indicate that hystricomorpha may have separated from myomorpha and castorimorpha before the separation of hystricomorpha from lagomorpha.

Medium-term effects of the diluted pig semen irradiation with red LED light on the integrity of nucleoprotein structure and resilience to withstand thermal stress

This study sought to evaluate the effects of irradiating pig seminal doses with red LED light irradiation on their quality and longevity over liquid-storage at 17 °C. For this purpose, boar ejaculates were diluted in a commercial extender at a final concentration of 3 × 107 sperm/mL and stored at 17 °C for 96 h. Upon arrival to our laboratory (5–6 h within collection), 1.5 mL-aliquots were subjected to irradiation with a temperature-controlled red light-emitting diode (LED) for 1 min, 5 min or 10 min. Controls consisted of non-irradiated spermatozoa. Aliquots were then stored at 17 °C for 96 h, and plasma membrane and acrosome integrity, motility and free cysteine radicals of sperm head proteins were evaluated every 24 h. In addition, the sperm resilience to withstand thermal stress following irradiation was evaluated at 24 h, 48 h, 72 h and 96 h by incubating stored seminal doses at 37 °C for 120 min. In our experimental conditions, light-stimulation for 5 min and 10 min counteracted the decrease in thermal stress observed in non-irradiated samples during the first 48 h of storage. Moreover, all irradiation protocols counteracted the decrease in percentages of spermatozoa with altered acrosomes observed in non-irradiated samples after 72 h of storage. The effects of light-stimulation upon sperm motility parameters were less consistent. While liquid-storage also led to an increase in the free cysteine levels of sperm head proteins, this increment was partially mitigated through light-stimulation for 5 min and 10 min. Our results suggest that effects linked with red LED light irradiation would be consistently maintained in our experimental conditions for the first 48 h. Finally, the maintenance of light effect appears to depend upon the specific experimental design, the analyzed sperm parameters and the utilized irradiation patterns.

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 150μM 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 5days. The BTS diluent containing RA improved the sperm quality during the process of liquid preservation compared with the control treatment. After 5days of liquid preservation, the addition of RA at 100μM 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 100μM 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.

Proteomics in fresh and preserved pig semen: Recent achievements and future challenges

Proteins in semen, either in spermatozoa (SPZ) or seminal plasma (SP), are directly involved in molecular processes and biological pathways regulating sperm function, including fertilizing ability. Therefore, semen proteins are candidates of choice for biomarkers discovery for fertility and for sperm (dys)function. Mass spectrometry (MS)-based proteomics has opened up a new era for characterizing and quantifying the protein profile of SP and SPZ, as well as for unveiling the complex protein interactions involved in the activation of sperm functionality. This article overviews existing literature on MS-based proteomics regarding porcine semen, with a specific focus on the potential practical application of the results achieved so far. The weaknesses of current studies and the perspectives for future research in MS-based pig semen proteomics are also addressed.

Effect of L-carnitine on sperm quality during liquid storage of boar semen.

ObjectiveThis study was conducted to investigate the effect of L-carnitine on the pig semen characteristics during storage.MethodsSpermatozoa samples were examined for spermatozoa quality and then randomly divided into 5 groups: 0 (control), 12.5, 25, 50, and 100 mM L-carnitine. Sperm motility, plasma membrane integrity and antioxidant parameters (total reactive oxygen species, total antioxidant capacity, and malondialdehyde) were evaluated after 0, 3, 5, and 10 day cooled-storage at 17°C. Moreover, ATP content, mitochondria activity as well as sperm-binding and in vitro fertilizing ability of preserved boar sperm were also investigated.ResultsSupplementation with 50 mM L-carnitine could effectively maintain boar sperm quality parameters such as sperm motility and membrane integrity. Besides, we found that L-carnitine had positive effects on boar sperm quality mainly through improving antioxidant capacities and enhancing ATP content and mitochondria activity. Interestingly, by assessing the effect of L-carnitine on sperm fertility and developmental potential, we discovered that the extender containing L-carnitine could improve sperm quality and increase the number of sperms bounding to zona pellucida, without improving in vitro fertility and development potential.ConclusionThese findings suggested that the proper addition of L-carnitine to the semen extender improved boar sperm quality during liquid storage at 17°C.

Penambahan Bovine Serum Albumin pada Beltsville Thawing Solution Dapat Mempertahankan Kualitas Semen Babi yang Disimpan pada 15°C (ADDITION OF BOVINE SERUM ALBUMIN TO BELTSVILLE THAWING SOLUTION COULD MAINTAIN QUALITY OF PIG SEMEN STORED AT 15OC)

This study aims to maintain the quality of pig semen for longer during storage at 15oC, in an effort to support artificial insemination programs with the addition of Bovine Serum albumin (BSA) to diluent Beltsville Thawing Solution (BTS). This study uses a completely randomized design with five treatment groups, each To = semen was diluted with BTS without the addition of BSA ; T1 = with the addition of 5 mg BSA/mL diluent; T2 = with the addition of 10 mg BSA/mL diluent; T3 = with the addition of 15 mg BSA/mL diluent; T4 = with the addition of 20 mg BSA/mL diluent. Each treatment was repeated five times so that the number of samples used was twenty-five. The diluted cement is stored at 15oC for 72 hours then observing the quality of cement includes: progressive motility, dead spermatozoa, abnormalities, and intact plasma membranes. The data obtained were analyzed by analysis of variance, if there were differences followed by Duncan’s test. The results showed, addition of BSA concentration of 10 mg/mL and 15 mg/mL of diluent gives the same effect on the quality of cement during storage and significantly better (p &lt;0.05) when compared to the addition of 0 mg/mL, 5 mg/mL and 20 mg/mL diluents. It can be concluded, the addition of BSA 10 mg/mL BTS diluents can maintain the most optimal quality of pig semen against progressive motility, dead spermatozoa, abnormalities and intact plasma membranes.