Effect of astaxanthin in extenders on sperm quality and functional variables of frozen-thawed boar semen.

The aim of the study was to determine whether the presence of astaxanthin (ASX) protects boar spermatozoa against damage related to cryopreservation. Pooled ejaculates extended in Beltsville Thawing Solution (BTS) were used. Three experiments were conducted: 1) sperm samples were pre-incubated overnight (17 °C) with ASX (0, 0.5, 5, 15 µM) prior to freezing and then frozen using cooling and thawing extenders supplemented with ASX (0, 0.5, 5, 15 µM); 2) sperm samples were treated with ASX (0, 0.5, 5, 15 µM) only during overnight pre-incubation (17 °C) prior to cryopreservation; and 3) a thawing extender was supplemented with ASX (0, 0.5, 5, 15 µM). The groups were as follows: control (C; no treatment), ASX 1 (0.5 µM), ASX 2 (5 µM) and ASX 3 (15 µM). Total (TM) and progressive (PM) motility was analyzed using CASA, while sperm viability, reactive oxygen species generation, lipid peroxidation and apoptoticlike changes were analyzed using flow cytometry. Sperm variables were evaluated prior to freezing as well as 30 and 150 min after thawing. In Experiment 1, the values of TM and sperm viability post-thaw were less in the ASX 3 than C group. In Experiment 2, there was no effect of ASX on any of the sperm variables evaluated, while in Experiment 3, apoptotic-like changes were less in the ASX 1 than C group. In conclusion, there was a subtle beneficial effect on cryopreserved boar spermatozoa after addition of ASX to thawing media.

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.

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.

Breed differences of bull frozen-thawed semen.

The objective of this study was to investigate the quality of frozen-thawed semen from different bull breeds. Commercial frozen-thawed bull semen samples (26 per breed, 130 totally) of five breeds (Holstein [Η], Brown Swiss [BS], Limousin [L], Belgian Blue [BB], Blonde d' Aquitaine [BA]) were used. After thawing, each semen sample was subjected to thermal resistance test (TR) for 0.5 and 1 hr at 38°C and hypo-osmotic swelling test (HOST) for 1 hr at 150 mOsm at 37°C. Additionally, all samples were evaluated at times 0 hr (thawing), 0.5 hr (TR), 1 hr (TR) for kinetics by CASA [progressive, immotile, rapid, medium, slow moving spermatozoa, curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity (LIN), straightness (STR), beat cross-frequency (BCF), amplitude of lateral head displacement (ALH), wobble (WOB)]. Moreover, directly after thawing, all semen samples were evaluated for morphometry, morphology, viability and DNA fragmentation. Statistical analysis was conducted using a mixed model for repeated measures. The results showed (a) higher VCL after thawing in H, L breeds compared to BB and BA, (b) higher VAP after thawing in L compared to BB, BA, (c) higher values of progressive spermatozoa after TR in H, BS compared to BB, BA, (d) higher values of rapid spermatozoa after thawing and 0.5 hr of TR in H, BS, L compared to BB, BA, (e) lower viability in BA after thawing compared to H, BS, BB, (f) lower morphological abnormalities in H compared to L, BB, (g) higher head length in Η compared to BB. No significant differences were observed in the results from HOST and DNA fragmentation between breeds. In conclusion, quality characteristics of frozen-thawed bull semen are dependent on the breed. Frozen semen from BB and BA breeds should be handled more carefully after thawing, as it is more sensitive to stress.

Selectivity of porcine zona pellucida to bind spermatozoa with normal chromatin structure.

This study aimed to investigate the selective ability of swine zona pellucida (ZP) to bind spermatozoa with normal nuclear chromatin. Ten ejaculates of four boars were used, while hemizona assay was applied for evaluation of binding capability. The results of this study showed that swine ZP has the ability to select spermatozoa with normal chromatin structure for sperm-zona binding process.

Relationship between sperm quality traits and field-fertility of porcine semen.

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 x 10(8) sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 x 10(6) sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 x 10(9) spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2 to approximately 76.5%) and SCI (0.16 to approximately 4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity.

Effect of ram age on structural and functional competence of frozen-thawed spermatozoa in dairy sheep.

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen-thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1-2 years (young) or 4-5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of >or= 2.5 x 10(9) sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell medium and frozen in 0.25 ml straws. The end points of post-thawing semen evaluation were computer-assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per-cell analysis of lipid peroxidation using C11-BODIPY(581/591), sperm-hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen-synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non-capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = -0.63 to -0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.

Study on the in vitro effect of zearalenone and alpha-zearalenol on boar sperm-zona pellucida interaction by hemizona assay application.

The mycotoxin zearalenone (zen) impairs fertility in farm animals. The aim of the present study was to investigate the effect of zearalenone and its major metabolite (alpha-zearalenol) on boar semen binding capacity, under in vitro conditions. Extended boar semen was exposed to three different concentrations of zen and alpha-zen (40, 60 and 80 microg ml(-1) of semen) for 1 h. Afterwards, the semen was washed and incubated with homologous oocyte hemizona for 4 h. A significant decrease (P < 0.001) in the number of tightly attached spermatozoa on the hemizona was obtained at concentrations of 60 microg ml(-1) and 80 microg ml(-1) of zen and alpha-zen. In conclusion, zen and alpha-zen affected the sperm-zona interaction by reducing the ability of boar spermatozoa to bind to the zona pellucida.

In vitro effect of zearalenone and alpha-zearalenol on boar sperm characteristics and acrosome reaction.

This study was conducted to determine the in vitro effects of three different concentrations (125, 187.5 and 250 microM in diluted semen) of zearalenone (zen) and alpha-zearalenol (alpha-zen) on boar sperm. Semen parameters such as motility, viability and spontaneous acrosome reaction were evaluated. From the results it was shown that both zen and alpha-zen affected the sperm characteristics significantly (p < 0.05), except for alpha-zen at the low concentration which did not decrease the percentage of live reacted spermatozoa significantly. In conclusion, zen and alpha-zen are directly toxic when they affect boar semen in vitro and consequently decrease the fertilization ability of the sperm. The higher the concentration of mycotoxin tested, the greater the decline of sperm parameters noticed. The influence of mycotoxins was found to be time- and dose-dependent.