Effects of Bovine Serum Albumin on Boar Sperm Quality During Liquid Storage at 17°C

This study aimed to investigate the effects of bovine serum albumin (BSA) on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 2, 3, 4, 5 and 6 g/l) of BSA, and sperm motility, plasma membrane integrity, acrosome integrity, total antioxidative capacity (T‐AOC) activity and malondialdehyde (MDA) content were measured and analysed. The results showed that Modena supplemented with 3, 4 and 5 g/l BSA could improve boar sperm motility, effective survival time and plasma membrane integrity (p < 0.05), decrease MDA content (p < 0.05), while no statistical difference was observed for sperm acrosome integrity and T‐AOC activity among these three groups (p > 0.05). The semen sample diluted with Modena containing 4 g/l BSA could achieve optimum effect, and sperm survival time was 7.5 days. After 7 days preservation, sperm motility, plasma membrane integrity and acrosome integrity were 54%, 49% and 78%, respectively. T‐AOC activity and MDA content were 1.03 U/ml and 17.5 nmol/ml, respectively. In conclusion, Modena supplemented with BSA reduced the oxidative stress and improved the sperm quality of boar semen during liquid storage at 17°C, and 4 g/l BSA was the optimum concentration. Further studies are required to obtain more concrete results on the determination of antioxidant capacities of BSA in liquid preserved boar semen.     

The effects of different levels of superoxide dismutase in Modena on boar semen quality during liquid preservation at 17°C

This study was conducted to investigate the influence of superoxide dismutase (SOD) on the quality of boar semen during liquid preservation at 17°C. Semen samples from 10 Duroc boars were collected and pooled, divided into five equal parts and diluted with Modena containing different concentrations (0, 100, 200, 300 and 400 U/mL) of SOD. During the process of liquid preservation at 17°C, sperm motility, acrosome integrity, membrane integrity, total antioxidant capacity (T‐AOC) activity, malondialdehyde (MDA) content and hydrogen peroxide (H₂O₂) content were measured and analyzed every 24 h. Meanwhile, effective survival time of boar semen during preservation was evaluated and analyzed. The results indicated that different concentrations of SOD in Modena showed different protective effects on boar sperm quality. Modena supplemented with SOD decreased the effects on reactive oxygen species on boar sperm quality during liquid preservation compared with that of the control group. The added 200 U/mL SOD group showed higher sperm motility, membrane integrity, acrosome integrity, effective survival time and T‐AOC activity. Meanwhile, the added 200 U/mL SOD group showed lower MDA content and H₂O₂content. In conclusion, addition of SOD to Modena improved the boar sperm quality by reducing oxidative stress during liquid preservation at 17°C and the optimum concentration was 200 U/mL.     

Supplementation of salvianic acid A to boar semen extender to improve seminal quality and antioxidant capacity

The purpose of this test was to investigate the effect of salvianic acid A (SAA, CAS No. 76822‐21‐4) on the quality of boar semen during liquid storage at 17°C. The effects of different concentrations of SAA on semen quality and antioxidant capacity were analyzed. Boar semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations (0, 15, 30, 45, 60, 75 μM of SAA). During the storage period, sperm activity was measured every 24 hr, and plasma membrane integrity, acrosome integrity, total antioxidant capacity (T‐AOC), malondialdehyde (MDA) content, and catalase (CAT) activity were measured at 0, 1, 3, and 5 days. The results from our study suggest that different concentrations of SAA have different effects on semen preservation. Semen samples supplemented with SAA showed reduced effects of oxidative stress on sperm compared to the control samples. Supplementation of 30 μM of SAA significantly improved sperm motility, plasma membrane integrity, acrosome integrity, and antioxidant capacity. However, the addition of SAA to the extender was scarcely beneficial to the improvement of results of artificial insemination with boar semen after liquid preservation. Further studies are necessary in order to demonstrate that SAA has good effects on the liquid preservation of semen.     

Protective influence of rosiglitazone against time‐dependent deterioration of boar spermatozoa preserved at 17°C

Spermatozoa are highly specialized cells, and energy metabolism plays an important role in modulating sperm viability and function. Rosiglitazone is an antidiabetic drug in the thiazolidinedione class that regulates metabolic flexibility and glucose uptake in various cell types, but its effects on boar sperm metabolism are unknown. In this study, we investigated the potential effect of rosiglitazone against time‐dependent deterioration of boar spermatozoa during liquid preservation at 17°C. Freshly ejaculated semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations of rosiglitazone, and the motility, membrane and acrosome integrity of sperm were detected. Besides, we measured glucose uptake capacity, l ‐lactate production level, mitochondrial membrane potential, adenosine triphosphate (ATP) content and mitochondrial reactive oxygen species (mROS) production of sperm after boar semen had been incubated with or without rosiglitazone, iodoacetate (glycolysis inhibitor) and rotenone (electron transport chain inhibitor) for 5 days. The addition of rosiglitazone significantly enhanced sperm quality and had a strong protective effect on the sperm membrane and acrosome integrity during storage. BTS containing 50 μM rosiglitazone maintained the total motility of liquid‐preserved sperm above 60% for 7 days. Rosiglitazone improved sperm quality by regulating energy metabolism manner of preserved sperm, protected the sperm mitochondrial membrane potential, enhanced sperm ATP production and in the meanwhile reduced mROS through enhancing glycolysis but not oxidative phosphorylation. The data suggested the practical feasibility of using rosiglitazone for improving boar spermatozoa quality during semen preservation.     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.     

Breed of Boar Influences the Optimal Concentration of Gamma‐oryzanol Needed for Semen Cryopreservation

The aim of this study was to investigate the influence of boar breed on the optimal concentration of gamma‐oryzanol on the qualities of cryopreserved boar semen. Semen was collected from 20 boars (10 Duroc, 5 Large white and 5 Landrace boars). The semen sample was divided into five groups (A–E) according to the concentration of gamma‐oryzanol in extender II, that is 0, 0.08, 0.16, 0.24 and 0.32 mm , respectively. The semen was cryopreserved by nitrogen vapour and storage in nitrogen tank (?196°C). After storage for a week, samples were thawed at 50°C for 12 s and evaluated for progressive motility, sperm viability and acrosome integrity. The results demonstrated that gamma‐oryzanol significantly improved progressive motility, viability and acrosome integrity of frozen–thawed boar semen. Considering the influence of breeds on the optimal concentration of gamma‐oryzanol, for Duroc boar, gamma‐oryzanol at 0.16 mm (group C) yielded the highest percentage of progressive motility, sperm viability and acrosome integrity. For Large white and Landrace boars, gamma‐oryzanol at 0.24 mm (group D) showed a significantly higher percentage of progressive motility, viability (not significant in Landrace) and acrosome integrity than other concentrations. In conclusion, the optimal concentration of gamma‐oryzanol needed for boar semen cryopreservation in lactose–egg yolk (LEY) freezing extender is not only depended on individual boar but also breed of boar, that is 0.16 mm for Duroc and 0.24 mm  for Large white and Landrace.     

Comparative Study on Five Different Commercial Extenders for Boar Semen

Increasing interest in a longer preservation of diluted boar sperm raises questions in the field concerning the choice of the extender. The aim of this study was to evaluate the longevity of boar sperm extended in currently used commercial semen extenders. Three long-term extenders and two short-term extenders were compared for different semen quality parameters that can be assessed under routine laboratory conditions. Sperm morphology, motility, pH and bacteriological contamination were investigated during a 7-day period. The number of dead spermatozoa did not differ significantly among the extenders (p > 0.05). Sperm motility was not only related with storage period but most of all with pH, especially in long-term extenders. Differences between the different extenders were prominent (p < 0.05); the sperm preserved in only one long-term extender showed good motility during the whole test period. In all cases, the pH of the extended semen increased by 0.3-0.5 in the first days of storage and was significantly correlated with a decrease in motility. Bacteriological quality had no significant influence on motility or pH of the semen. In conclusion, we can state that in both short-term extenders and in only one long-term extender, sperm longevity, as evaluated by the parameters used in this study, was sufficient during the preservation period. To preserve the quality of diluted boar semen during long-term storage, the choice of the long-term extender is important. In addition, the monitoring of the pH of extended boar semen in our study emphasizes the importance of the buffering capacity of semen extenders.     

猪精液超低温冷冻保存研究进展

猪人工授精(AI)技术已经广泛应用于畜牧业生产,但精液来源基本是17℃保存的新鲜猪精液,不利于精液长期保存和猪种资源保存。超低温冷冻保存技术是猪精液长期保存最有效的方法,冷冻精液可以实现不同地区、不同国家优良种猪精液间的交流,从而大大提高猪种资源的利用率,但冷冻解冻后的精液质量较差,受胎率、窝产仔数远低于鲜精,极大地限制了猪精液冷冻保存技术的广泛应用。本文综述了猪精液冷冻保存研究概况、猪精液冷冻损伤机理和猪精液冷冻保存影响因素,以期为猪精液超低温冷冻保存研究提供新的思路和方法。     

Notoginsenoside R1 protects boar sperm during liquid storage at 17°C

Reactive oxygen species (ROS) damage mammalian sperm during liquid storage. Notoginsenoside R1 (NR1) is a compound isolated from the roots of Panax notoginseng; it has powerful ROS-scavenging activities. This work hypothesized that the antioxidant capacity of NR1 could improve boar sperm quality and fertility during liquid storage. During liquid storage at 17°C, the supplementation of semen extender with NR1 (50 μM) significantly improved sperm motility, membrane integrity and acrosome integrity after 5 days of preservation. NR1 treatment also reduced ROS and lipid peroxidation (LPO) levels at day 5 (p <0.05). Higher glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) levels and sperm–zona pellucida binding capacity were observed in the 50 μM NR1 group than those in the control group at day 7 (p <0.05). Importantly, statistical analysis of the fertility of 200 sows indicated that addition of NR1 to the extender improved the fertility parameters of boar spermatozoa during liquid storage at 17°C (p <0.05). These results demonstrate the practical feasibility of using 50 μM NR1 as an antioxidant in boar extender during liquid storage at 17°C, which is beneficial to both spermatozoa quality and fertility.     

Effects of iodine methionine on boar sperm quality during liquid storage at 17°C

Microbial environment is one of the important factors that affect the quality of preserved semen. Iodine methionine (IM), participating in the production and activation of metabolic enzymes, is a new type of amino acid chelate. To date, there has been no report to evaluate the effects of IM on boar semen preservation at 17°C. This study was designed to investigate the effects of IM on boar sperm quality and reproductive performance during liquid storage at 17°C and its antibacterial effect. Semen samples collected from six Yorkshire boars were diluted with basic liquid containing different concentrations of IM (0, 20, 40, 80, 160 and 320 μM). Subsequently, sperm motility, plasma membrane integrity and acrosome integrity were determined. After 6 days of preservation, the difference in microbial composition between control group and 80 μM IM group was compared using 16S rDNA sequencing, and the effects of IM on reproductive performance were also compared and analysed between the two groups. The results demonstrated that 20, 40 and 80 μM IM improved boar sperm motility, plasma membrane integrity and acrosome integrity. 80 μM IM was the optimum concentration. Conversely, 160 and 320 μM IM resulted in deleterious consequences to boar sperm quality compared to the control group and other treatment groups (p < .05). After 6 days of preservation, sperm motility, plasma membrane integrity and acrosome integrity were 56.0%, 51.8% and 59.4%, respectively. There was no significant difference in non‐return rate between the two groups (p > .05). But the litter size of 80 μM IM group was significantly higher than that of control group (p < .05). 80 μM IM inhibited proliferation of the phylum Proteobacteria and the genus Staphylococcus as well as Pseudomonas (p < .05). Further studies are required to understand the antibacterial mechanism of IM in liquid‐preserved boar semen.     

Analysis of In vitro Fertilizing Capacity to Evaluate the Freezing Procedures of Boar Semen and to Predict the Subsequent Fertility

A porcine in vitro fertilization (IVF) system and seminal quality parameters of frozen–thawed boar semen were used to assess the effectiveness of two different thawing rates of frozen boar semen, and to address the question of whether differences between fertility of ejaculates could be predicted in a limited field trial. In the first experiment, two thawing procedures were analysed (37°C, 30 s; 50°C, 12 s) and no differences in sperm quality were found. However, when the procedure was 50°C, 12 s the IVF results showed a higher number of sperm per penetrated oocyte and a near 10 points higher rate of pronuclear formation. In the second experiment, the fertility results obtained in the limited field trial show to be efficient enough for application in a commercial use, especially for three of the employed boars (fertility ≥80%). In this limited study, the conventional seminal parameters are not accurate enough to discriminate good and bad boars in relation to fertility. On the contrary, parameters of in vitro penetrability are more precise to predict subsequent fertilities. As conclusion, the IVF fertilization system seems to be a good tool to evaluate the quality of frozen–thawed boar semen previous to its commercial way, to verify the bank semen storage quality and a good way to assay new sperm freezing procedures, as it is the more precise evaluating method in estimating the potential fertilizing ability.     

Effects of glutathione on sperm quality during liquid storage in boars

The aim of this study was to investigate the effects of different concentrations of glutathione in Modena on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 5, 10, 15 mmol/L) of glutathione. Sperm motility, effective survival period, plasma membrane integrity, acrosome integrity, total antioxidant capacity (T‐AOC) activity, malondialdehyde (MDA) content and hydrogen peroxide (H₂O₂) content were measured and analyzed. The results showed that Modena supplemented with 1, 5 and 10 mmol/L glutathione improved sperm motility, effective survival period, plasma membrane integrity and T‐AOC, and decreased MDA content and H₂O₂ content. Meanwhile, the semen sample diluted with Modena containing 1 mmol/L glutathione achieved optimum effect, and effective survival period was 6.1 days. After 5 days preservation, sperm motility, plasma membrane integrity and T‐AOC of the group treated with 1 mmol/L glutathione were all higher than that of other groups. Meanwhile, MDA content and H₂O₂ content were lower than that of other groups. In conclusion, Modena supplemented with glutathione decreased the oxidative stress and improved the quality of boar semen during liquid storage at 17°C, and 1 mmol/L concentration was the optimum concentration. © 2016 Japanese Society of Animal Science     

Supplemental effect of different levels of taurine in Modena on boar semen quality during liquid preservation at 17°C

Peroxidation damage induces sublethal injury to boar sperm during the storage process. Taurine has already been demonstrated to protect cells effectively from oxidant‐induced injury. This study was aimed to evaluate the effect of different concentrations of taurine (0.5, 1, 5 and 10 mmol/L) in Modena diluent on boar sperm quality during liquid storage at 17°C. Ejaculates from sexually mature Duroc pigs were collected, pooled and preserved in the Modena containing different concentrations of taurine. Sperm motility, plasma membrane integrity, acrosome integrity, total antioxidative capacity (T‐AOC) activity and malondialdehyde content (MDA) were examined every 24 h. Modena diluent containing taurine suppressed the reduction in sperm qualities during the process of liquid preservation compared with those of the control group. After 5 days of liquid preservation, the addition of taurine at 5 mmol/L had the optimal effect on survival time as well as maintenance of motility, plasma membrane integrity, acrosomal integrity, T‐AOC activity and MDA content. These results may suggest the possibility that the proper addition of taurine to the semen extender improves the swine production system using artificial insemination by the suppressing of sperm damage and subsequent dysfunction during liquid preservation.     

Irradiation of semen doses with LED‐based red light in a photo chamber does not improve in vitro quality of thermically stressed boar spermatozoa

Recent reports indicate that stimulation of liquid‐stored boar semen with red LED‐based light improves sperm quality and reproductive performance in sow herds. So far, in vitro data after LED stimulation of whole semen doses are lacking. In this study, the effect of LED light exposure on the in vitro quality of boar spermatozoa after storage and thermic incubation was examined. Boar semen doses were stored at 17°C (n = 10) or 5°C (n = 6) in Beltsville Thawing Solution extender and then exposed to red LED light using a commercial photo chamber. During a subsequent long‐term incubation at 38°C, neither sperm kinematic parameters nor mitochondria function or membrane integrity differed between control and treated samples (p > .05). It is concluded that stimulation of semen doses in the LED‐photo chamber does not improve quality of thermically stressed boar sperm in vitro. Other than the sperm traits tested here might be involved in the previously reported improvement of in vivo fertility.     

猪精液常温保存抗氧化剂研究进展

猪的人工授精技术被广泛用于我国生猪养殖业。用于人工授精的公猪精液质量直接关系到人工授精后母猪的受胎率和产仔数。猪精子质膜中胆固醇/磷脂比值低致使其对冷应激十分敏感,这决定了常温保存(16~18℃)成为目前最常用的猪精液保存方法。猪精液常温保存过程会累积活性氧自由基,而猪精子质膜中多不饱和脂肪酸比例高,使精子极易受到氧化应激伤害。稀释液中添加抗氧化剂有助于维持精子质量的各项指标。抗氧化剂主要包括超氧化物歧化酶等酶类抗氧化剂和还原型谷胱甘肽等非酶类抗氧化剂。本文综述了猪精子的生物学特征和常温保存过程中猪精液累积活性氧的原因,并总结了猪精液常温保存中抗氧化剂的研究进展,旨在为猪精液的保存及人工授精提供参考。     

Evaluation of cryopreserved boar semen after supplementation sericin form silkworm (Bombyx mori) in semen extender

Boar cryopreserved semen is scarcely used for artificial insemination due to its quality which is largely reduced by membrane lipid peroxidation. This present study was designed to improve the post‐thawed boar semen quality by determining the optimal level of sericin supplementation (antioxidants) in semen extender. Five levels of sericin supplementation between 0% and 1% (w/v) were examined. Semen was frozen by the liquid nitrogen vapor method, thawed slowly at 5°C for 5 min, and used for the evaluation of sperm quality. The results indicated 0.5%–1% sericin supplementation was more effective on maintenance of sperm viability, acrosome integrity, and mitochondrial functions during freezing–thawing. Moreover, 0.75% sericin supplementation was most protective toward total sperm motility and sperm progressive motility. Additionally, 0.25%–0.75% sericin supplementation significantly suppressed increases in the index of lipid peroxidation. In conclusion, 0.75% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved boar semen. However, further research using AI will be necessary to demonstrate that this indication can be applied to the production of offspring in the farms.     

Factors influencing boar sperm cryosurvival

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.     

二甲基甲酰胺对猪精液冷冻保存效果的影响

用二甲基甲酰胺(DMF)完全替代甘油,比较不同平衡时间和不同DMF添加量对猪精液冷冻保护效果的影响。结果表明,DMF能完全替代甘油,获得较好的冷冻保护效果。最佳平衡时间为90 min,解冻后精子活力为(44.57±0.72)%,显著高于对照组和其他组(P0.05)。当DMF添加量为5%时,冻后精子活力、活率、线粒体活性、顶体完整率和质膜完整率分别为(49.91±0.39)%(、46.51±0.26)%、(47.51±0.52)%(、49.84±0.56)%、(46.30±1.61)%,均显著高于2%、3%、6%DMF添加组(P0.05),但与4%DMF添加组相比,冻后精子活力、活率和质膜完整率差异不显著(P0.05)。本试验结果表明,DMF最适添加量为5%。     

Assessment of storage effects in liquid preserved boar semen

Fertility of extended boar semen declines within the first 72 h of storage in vitro. Standard semen assessment, such as motility and membrane integrity, allows detection of lethal damage of spermatozoa. However, conventional sperm assessment often lacks standardization and does not allow identification of sub-lethal changes of sperm quality during the initial 72 h of storage. In the present brief review, recent strategies for quality assessment of liquid preserved boar semen are discussed and basic implications for experiments designed to detect storage effects are given.     

Effect of astaxanthin on the quality of boar sperm stored at 17°C,incubated at 37°C or under in vitro conditions

The aim of the study was to investigate the effect of the antioxidant astaxanthin on boar semen. Twenty ejaculates from 10 boars (two ejaculates/boar) were extended and split in three groups: semen control (SC), solvent control (C; semen with dimethyl sulfoxide, the diluent of astaxanthin) and semen with astaxanthin (A) in concentration 0.5 μmol/L. Sperm quality parameters (motility and kinetics, morphology, viability, functional integrity of sperm plasma membrane by Hypo‐Osmotic Swelling Test [HOST] and DNA integrity) were assessed at 0, 24 and 48 hr of storage at 17°C (experiment I), before (0 hr) and after (1 hr) of sperm thermal resistance assay at 37°C (experiment II) and finally before (0 hr) and after (1 hr) sperm in vitro incubation (38.5°C, 5% CO₂, maximum humidity [experiment III]). In experiment I, group A performed overall better than group SC and as a tendency better than group C regarding viability. Total motility, rapid spermatozoa and HOST remained constant across time in group A, whereas they decreased in the remaining groups. In experiment II, regarding motility and viability, group A displayed better results across time than the other two groups. In experiment III, viability and total motility decreased in groups SC and C, while in group A, these parameters were not significantly different between the examination time points. In conclusion, astaxanthin has a beneficial and protective effect on boar semen quality under the investigated conditions.