Spermatozoa in the sperm-peak-fraction of the boar ejaculate show a lower flow of Ca(2+) under capacitation conditions post-thaw which might account for their higher membrane stability after cryopreservation.

Boar spermatozoa collected in the ejaculate sperm peak-portion (P1, first 10 mL of the sperm-rich fraction, SRF), had shown a higher resilience to freezing and thawing compared to spermatozoa from the rest of the ejaculate (2nd portion of the SRF plus the post-sperm-rich fraction, PSRF), even when using a simplified freezing technique, as long as spermatozoa were incubated in their own seminal plasma (SP). This experiment studied the stability of P1- and SRF-P1 boar spermatozoa frozen in MiniFlatPacks (MFP), post-thaw, using flow cytometry. Since spermatozoa from either portion showed similar cryosurvival and low proportions of unstable membranes (<3%, annexin-V/propidium iodide staining), and only a tendency for SRF-P1 live spermatozoa to depict acrosome exocytosis (FITC-PNA/PI/H33342); they were explored for Ca(2+) contents using a Fluo-4 probe under in vitro capacitating conditions (mBO+ medium), as well they were tested for their ability to sustain a short Ca(2+)-ionophore (A23187) in vitro challenge. The proportions of live spermatozoa depicting high Ca(2+)-levels were initially <2% but increased over incubation time, particularly in SRF-P1(P<0.05), while proportions of live spermatozoa with low Ca(2+)-levels were basically constant over incubation time (~11-14%), for either portion. Incubation in capacitation medium did not modify the proportions of low-Ca(2+) but dramatically increased the proportions of high-Ca(2+) spermatozoa (P<0.001) already after 15 min exposure, highest for SRF-P1 spermatozoa. While the proportion of live spermatozoa with intact acrosome was significantly decreased among SRF-P1 (P<0.001), that of P1-spermatozoa remained unchanged, probably owing to the lowest relative content of cytosolic Ca(2+). The results suggest that spermatozoa in the P1-portion are more resilient to express acrosome exocytosis post-thaw compared to those bathing in the rest of the SRF-fraction when cryopreserved using a simplified technique, in MFPs.

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art.

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of 'bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.

Dietary Karaya Saponin and Rhodobacter capsulatus Exert Hypocholesterolemic Effects by Suppression of Hepatic Cholesterol Synthesis and Promotion of Bile Acid Synthesis in Laying Hens.

This study was conducted to elucidate the mechanism underlying the hypolipidemic action of karaya saponin or Rhodobacter (R.) capsulatus. A total of 40 laying hens (20-week-old) were assigned into four dietary treatment groups and fed a basal diet (as a control) or basal diets supplemented with either karaya saponin, R. capsulatus, or both for 60 days. The level of serum low-density-lipoprotein cholesterol and the levels of cholesterol and triglycerides in the serum, liver, and egg yolk were reduced by all the supplementations (P < .05). Liver bile acid concentration and fecal concentrations of cholesterol, triacylglycerol, and bile acid were simultaneously increased by the supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus (P < .05). The supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus suppressed the incorporation of (14)C from 1-(14)C-palmitic acid into the fractions of total lipids, phospholipids, triacylglycerol, and cholesterol in the liver in vitro (P < .05). These findings suggest that the hypocholesterolemic effects of karaya saponin and R. capsulatus are caused by the suppression of the cholesterol synthesis and the promotion of cholesterol catabolism in the liver.

Metabolism of exogenous fatty acids, fatty acid-mediated cholesterol efflux, PKA and PKC pathways in boar sperm acrosome reaction.

PURPOSE: For understanding the roles of fatty acids on the induction of acrosome reaction which occurs under association of cholesterol efflux and PKA or PKC pathways in boar spermatozoa, metabolic fate of alone and combined radiolabeled 14C-oleic acid and 3H-linoleic acid incorporated in the sperm was compared, and behavior of cholesterol and effects of PKA and PKC inhibitors upon fatty acid-induced acrosome reaction were examined. METHODS: Semen was collected from a Duroc boar, and the metabolic activities of fatty acids in the spermatozoa were measured using radioactive compounds and thin layer chromatography. Cholesterol efflux was measured with a cholesterol determination assay kit. Participation of fatty acids on the AR through PKA and PKC pathways was evaluated using a specific inhibitor of these enzymes. RESULTS: Incorporation rate of 14C-oleic acid into the sperm lipids was significantly higher than that of 3H-linoleic acid (P < 0.05). The oxidation of 14C-oleic acid was higher in combined radiolabeling rather than in one. The highest amounts of 3H-linoleic acid and 14C-oleic acid were recovered mainly in the triglycerides and phospholipids fraction, and 14C-oleic acid distribution was higher than the 3H-linoleic acid in both labeled (P < 0.05) sperm lipids. In the 3H-linoleic and 14C-oleic acid combined radiolabeling, the incorporation rate of the radioactive fatty acids in all the lipid fractions increased 15 times more than the alone radiolabeling. Boar sperm utilize oleic acid to generate energy for hyperactivation (P < 0.05). Supplementation of arachidonic acid significantly increased (P < 0.05) cholesterol efflux in sperm. When spermatozoa were incubated with PKA or PKC inhibitors, there was a significant reduction of arachidonic acid-induced acrosome reaction (AR) (P < 0.05), and inhibition by PKA inhibitor is stronger than that by PKC inhibitor. CONCLUSIONS: Incorporation of unsaturated fatty acids, especially oleic acid, into triglycerides and phospholipids provides prerequisite energy for AR. Cholesterol efflux by arachidonic acid triggers AR. Arachidonic acid activated PKA and PKC pathway participate in induction of the AR.

Effect of amino acids and dipeptides on the acrosome reaction and accumulation of ammonia in porcine spermatozoa.

Aim: The present study was designed to investigate the effect of amino acids and their dipeptides in the medium related to the urea cycle on the motility, viability, acrosome reaction (AR) and accumulation of ammonia in the medium over different incubation periods in porcine spermatozoa and to assess the utilization of glucose. Methods: Porcine spermatozoa were washed, swim-up and incubated at 37°C for 0-4 h in mTALP medium supplemented with 75-600 µmol/L ammonia. Amino acids (1.0 mmol) or their dipeptides (2.0 mmol) were added individually to the mTALP medium containing either no ammonia or 300 µmol/L of ammonia. The viability and AR of porcine spermatozoa were assessed using the triple-staining technique and the accumulation of ammonia in the medium was measured using the indophenol method. Results: The motility, viability and AR were adversely affected (P < 0.05) by concentrations of ammonia ≥300 µmol/L compared with the control. Supplementation of l-alanyl-l-glutamine (AlaGln), l-glycyl-l-glutamine (GlyGln) and AlaGln + GlyGln in the presence of 300 µmol/L ammonia significantly increase (P < 0.05) the rate of motility, viability, AR, incorporation, accumulation of ammonia and oxidation of 14C(U)-glucose compared with the ammonia supplement control. Conclusion: AlaGln and GlyGln in mTALP medium were more stable and effective than the individual amino acids in reducing the accumulation of ammonia, and subsequently increasing the rate of AR and the utilization of glucose in porcine spermatozoa. (Reprod Med Biol 2008; 7: 123-131).

Effect of fatty acids bound to bovine serum albumin-V on acrosome reaction and utilization of glucose in boar spermatozoa.

Aim: The present study has been designed with the objective of determining if fatty acids bound to bovine serum albumin-V (BSA-V) can improve motility, viability, and increase acrosome reaction (AR) and utilization of glucose in boar spermatozoa. Methods: Boar spermatozoa were washed, swum-up and incubated at 37°C for 6 h in TALP medium supplemented with fatty acids bound to bovine serum albumin fraction V (BSA-V), fatty acid free BSA (BSA-FAF), polyvinyl alcohol + main fatty acids bound to BSA-V (PVA + FA) and PVA. Sperm motility, viability, AR, and the incorporation and oxidation of 14C-glucose were evaluated during 6 h of incubation. Results: The results show that the BSA-V was superior to BSA-FAF and PVA in improving motility and AR. Viability was significantly increased (P < 0.05) by only BSA-V compared with PVA. When the main fatty acids compound of BSA-V were added to PVA, the sperm motility, viability and AR became almost the same as with BSA-V. The rate of incorporation and oxidation of 14C-glucose were significantly increased (P < 0.05) by BSA-V compared with BSA-FAF and PVA. Fatty acids bound to BSA-V are important for improvement of sperm functions. Conclusions: The present study postulates that fatty acids bound to BSA-V are important to acrosome reaction and the utilization of glucose in boar spermatozoa.

Effect of fatty acids on boar sperm motility, viability and acrosome reaction.

Aim: The present study was undertaken to determine which fatty acids improve motility, viability, and increase acrosome reaction (AR) in boar spermatozoa. Methods: Boar spermatozoa were washed, swum-up and incubated at 37°C for 4 h in TALP medium supplemented with myristic, palmitic, stearic, lignoceric, oleic, linoleic, arachidonic, docosahexaenoic and palmitoleic acid. Sperm motility, viability and AR were evaluated during 4 h of incubation. Results: Results show that oleic and linoleic acid significantly improved (P < 0.05) the motility and viability of boar spermatozoa. The AR was significantly improved (P < 0.05) by oleic and arachidonic acid in almost all incubation periods. When combinations of oleic, linoleic and arachidonic acid were studied for motility, viability and AR, it was found that oleic plus linoleic acid significantly increased (P < 0.05) motility, whereas arachidonic plus oleic acid significantly increased (P < 0.05) AR. Conclusion: Unsaturated fatty acids, especially arachidonic acid, can improve boar sperm motility and AR. A combination of arachidonic and oleic acid is important for inducing boar sperm AR. (Reprod Med Biol 2007; 6: 235-239).

Effect of relaxin on motility, acrosome reaction and viability of cryopreserved boar spermatozoa.

Background and Aims: Relaxin has an important role in stimulating motility and the acrosome reaction (AR) of fresh boar spermatozoa. The objective of the present study was to determine whether relaxin can improve the motility, AR and viability of cryopreserved boar spermatozoa. Methods: Cryopreserved boar spermatozoa were thawed, washed and incubated at 37°C for 4 h in modified Beltsville thawing solution supplemented with 0, 20 or 40 ng/mL relaxin. Sperm motility, AR, viability, and incorporation and oxidation of 14C-glucose were evaluated during 0-4 h of incubation. Results: The results show that the supplementation of relaxin (especially at 20 ng/mL) in the thawing solution improved sperm motility significantly (P < 0.05) at 1-3 h of incubation. The percentage of acrosome reacted live spermatozoa was improved significantly (P < 0.05) when the spermatozoa were treated with 20 ng/mL relaxin. Viability was not significantly (P > 0.05) improved by supplementation with relaxin. The rates of incorporation and oxidation of 14C-glucose were increased in correlation with AR up to 4 h of incubation. Conclusion: We conclude that relaxin can improve the sperm motility and AR, and enhance the glucose metabolism of cryopreserved boar spermatozoa. (Reprod Med Biol 2006; 5: 215-220).