Impact of changes in composition of storage medium on lipid content and quality of turkey spermatozoa

Turkey semen quality is damaged by long term in vitro storage. The objective of the present study was to determine whether changes in energy substrates and antioxidants of semen extender could limit loss of quality and lipid content of turkey spermatozoa during storage. Spermatozoa were incubated in extenders based on Beltsville Poultry Semen Extender (BPSE) to which different energy substrates (acetate, pyruvate and hydroxybutyric acid) or antioxidant (Vitamin E) had been added. Semen was stored at 4 degrees C for 48 h and changes in quality, phospholipid and malondialdehyde (MDA) content of semen were evaluated. Among the different substrates studied, only acetate was able to limit the loss of motility and ATP content after 48 h in vitro storage. Losses of spermatozoal phospholipids were similar when gametes were incubated in an extender without any substrate or in normal BPSE (784-675nmol/10(9) spz versus 837-703 nmol/10(9) spz). However, motility and ATP content were significantly more affected after 48 h of storage in samples incubated without substrates than in BPSE (motility, 2.2 versus 0; ATP, 10 nmol/10(9) spz versus 3 nmol/10(9) spz). The addition of Vitamin E to the extender did not modify either the MDA or phospholipid content of fresh or stored spermatozoa, but increased the motility of stored semen. In conclusion, acetate is an essential substrate for in vitro storage. Spermatozoal phospholipids decreased during storage, but this did not seem to originate from metabolism of endogenous fatty acids. The positive effects of Vitamin E on semen storage did not originate from preservation of lipid oxidation.     

Liquid storage of turkey semen: changes in quality parameters, lipid composition and susceptibility to induced in vitro peroxidation in control, n-3 fatty acids and alpha-tocopherol rich spermatozoa

The study considered two major aims: (a) to measure the changes in quality parameters, lipid composition and antioxidant activity occurring in turkey spermatozoa during liquid storage; (b) to determine if the enrichment of sperm in n-3 fatty acids and alpha-tocopherol affect sperm survival during storage. Turkey breeders were fed a control diet or an Omega3 diet enriched with fish oil and alpha-tocopheryl-acetate. Ejaculates were pooled (5ejaculates/pool; 4pools/treatment) and stored in vitro for 48h at 4 degrees C. Viability, motility, susceptibility to induced peroxidation and alpha-tocopherol content were measured in spermatozoa; lipid and phospholipid fatty acid composition were measured in spermatozoa and seminal plasma. The proportion of motile and viable spermatozoa significantly decreased, and the proportion of dead spermatozoa significantly increased. The susceptibility of turkey spermatozoa to induced peroxidation also significantly increased during storage. The enrichment of turkey spermatozoa with n-3 long chain PUFA and vitamin E by dietary treatment did not prevent the negative effect of storage on sperm quality and sensitivity to induced in vitro peroxidation; however, it was efficient in partially prevent the increase of sperm death, therefore the proportion of dead spermatozoa was higher in control (37.4%) compared to treated spermatozoa (31.7%) after 48h liquid storage. Major changes were recorded in the lipid composition of turkey spermatozoa during liquid storage in both experimental dietary groups, whereas no significant changes were measured in seminal plasma. In spermatozoa, a great loss in the phospholipid and free cholesterol content was measured. Moreover, the loss in total sperm phospholipid was associated to a peculiar and selective decrease in the bounded fatty acids: saturates and monounsaturates were greatly reduced and polyunsaturates did not change. As a consequence, the polyunsaturated to saturated fatty acid ratio increased during 48h liquid storage. The observed changes in the lipid and phospholipid-bound fatty acid composition of turkey spermatozoa occurring during liquid storage might be related to different events and have been discussed.     

Changes in turkey semen lipids during liquid in vitro storage

The changes in lipid composition of spermatozoa and seminal plasma and changes in motility, viability, and morphological integrity of spermatozoa were measured in turkey semen diluted in Beltsville poultry semen extender and stored for 48 h (4 degrees C). The total phospholipid content of spermatozoa decreased during storage, while no quantitative decrease was observed in seminal plasma. More precisely, significant decreases in phosphatidylcholine, and to a lesser extent in sphingomyeline, phosphatidylserine, and phosphatidylinositol were observed in spermatozoa. The fatty acid profile of turkey spermatozoa partly reflected diet composition and had a high level of n-9 polyunsaturated fatty acids. Neither fatty acid profile nor free cholesterol were affected by storage. The lipid composition of seminal plasma was quite different from that observed in spermatozoa and was similar to the high density lipoprotein composition of chicken seminal plasma. In vitro storage did not significantly affect lipid classes and only small changes were observed in phospholipid classes of seminal plasma. The motility, viability, and morphological integrity of spermatozoa decreased during storage. These changes in phospholipid content may be explained by membrane phospholipid lysis followed by endogenous metabolism or by a complex combination of lysis, metabolism, and peroxidation. They are likely to affect semen quality and the success of in vitro storage severely.     

Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in Turkeys

Semen of Turkeys between 31 and 52 weeks of age was analyzed to investigate the cause of reduction in Turkey fertility at the end of the reproductive period. Sperm motility and viability, lipid concentration, fatty acid composition and lipid peroxides were evaluated on fresh spermatozoa or spermatozoa stored for 48h at 4 degrees C. Fertility of fresh semen was also evaluated.Fertility obtained with fresh semen decreased at 44-47 weeks of age. Ageing was also accompanied by a decrease in sperm viability (at 47 weeks) and later by a decrease in motility of spermatozoa (at 52 weeks). Polyunsaturated fatty acids (PUFAs) were the first lipids of fresh spermatozoa affected by age, especially n-3 and n-9 PUFAs. Changes in these PUFAs were followed by a 30% increase in lipid peroxidation at 47 and 52 weeks of age and a reduction in phospholipid content at 52 weeks.In vitro storage did not cause lipid peroxidation in sperm obtained during the first half of the reproductive period but malondialdehyde (MDA) levels significantly increased in sperm obtained during the second half of this period. In vitro storage also decreased phospholipid content of spermatozoa from 41 weeks of age, and viability and motility regardless of age.In conclusion, lipid alteration mainly originating from PUFAs peroxidation could partly explain the decrease in semen quality and fertility observed with ageing. In addition, lipid peroxidation was increased during in vitro storage of spermatozoa from older Turkeys.     

Effect of supranutritional level of dietary alpha-tocopheryl acetate and selenium on rabbit semen

This research examined the effects of dietary alpha-tocopheryl acetate (50 or 200 mg/kg diet) and selenium (Se, 0 or 0.5 ppm) supplementation on motion characteristics, oxidative stability and fertilizing ability of rabbit spermatozoa, fresh and stored for 24 h at 5 degrees C. The higher amount of dietary alpha-tocopheryl acetate increased the level of Vitamin E in the fresh semen (1.75 mmol/l versus 0.95 mmol/l) and its oxidative stability (thiobarbituric acid reactive substances-TBARS 12.44 nmol malondialdehyde/10(8) sperm versus 21.4 nmol malondialdehyde/10(8) sperm). Dietary Se increased gluthatione peroxidase activity (GPx) in erythrocytes (285 U/g Hb versus 207 U/g Hb), seminal plasma (270 U/l versus 190 U/l) and spermatozoa (1338 mU/10(9) sperm versus 1103 mU/10(9) sperm), whereas it did not show any effect on alpha-tocopherol level and TBARS. No synergy between Vitamin E and Se was shown. Storage for 24 h at 5 degrees C increased the TBARS level in all the experimental groups. Neither live and acrosome reacted spermatozoa, nor kinetic parameters, nor fertility rate were modified by dietary supplementation.     

Roles of antioxidants on prolonged storage of avian spermatozoa in vivo and in vitro

This review focuses on natural and assisted prevention against lipid peroxidation in avian spermatozoa. The presence of high levels of n-6 polyunsaturated fatty acids (PUFAs) in the plasma membrane creates favorable conditions for the formation of peroxidative products, a major cause of membrane damage which may ultimately impair male fertility. However, a complex antioxidant system involving vitamin C, vitamin E and GSH is naturally present in avian semen. Coupled with a battery of enzymatic defenses (e.g., SOD, GSH-Px either Se- or non-Se-dependent), this system acts to prevent or restrict the formation and propagation of peroxides. The presence of specialized sites dedicated to prolonged sperm storage in avian females raises the question of durable protection of sperm membranes against peroxidation. Preliminary observations have revealed the presence of a specific antioxidant system at these sites in which vitamin C could exert a major role. From a practical standpoint, the extensive use of artificial insemination in poultry, along with the emergence in some species of workable techniques to cryopreserve spermatozoa, demand better control of peroxidation occurring in the plasma membrane of spermatozoa before or during storage. Dietary supplementation with vitamin E is effective in limiting lipid peroxidation of sperm plasma membranes, both in chickens and turkeys. In addition, organic Se with or without vitamin E stimulates Se-GSH-Px activity in seminal plasma. Preliminary observations in female chickens have also revealed the effectiveness of dietary supplementation with vitamin E, organic selenium or both to sustain fertility in aging flocks.     

Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro.

This paper describes the effects of whole seminal plasma and of dialysed seminal plasma on the fertilizing ability of fowl spermatozoa stored for 24 h at 4 degrees C. The fertilizing ability of fowl semen diluted 1:1 with Beltsville Poultry Semen Extender and stored for 24 h at 4 degrees C was enhanced after replacement of the homologous seminal plasma by the diluent (89 versus 77% fertilization rate). Better results were obtained with seminal plasma dialysed against water before sperm storage to discard the less than 1 kDa or the less than 50 kDa fractions. It was concluded that low molecular weight seminal plasma fractions could damage the fertilizing ability of spermatozoa during storage at 4 degrees C, whereas high molecular weight fractions appeared to enhance fertilizing ability.     

Validation of a rapid, large-scale assay to quantify ATP concentration in spermatozoa

Quantification of ATP content in spermatozoa is a useful assay for evaluating sperm function; however, most detection methodology relies on assessing single samples. We have developed and validated a highly repeatable assay that permits simultaneous measurement of up to 78 samples. A key feature of this assay includes combination of a phosphatase inhibition and ATP extraction step that permits maximal detection of ATP and sample storage at -20 degrees C prior to assay. The assay was validated for spermatozoa from three different species, including turkey, rooster and boar. The sensitivity of the assay differed between avian and mammalian spermatozoa, with 2.5 x 10(6) spermatozoa being the lowest number of turkey and rooster spermatozoa that could be assayed compared to 2.5 x 10(5) boar spermatozoa. Concentrations of ATP in fresh turkey semen ranged from 2.14 to 15.6 nmol/10(9) spermatozoa; similarly, freshly collected rooster semen contained from 2.16 to 21.4 nmol ATP/10(9) spermatozoa. Evaluation of turkey semen that had been stored at 4 degrees C for 24 h revealed a decline in ATP concentrations (2.35 +/- 0.34 nmol ATP/10(9) spermatozoa). Likewise, cryopreserved rooster spermatozoa contained lower concentrations of ATP (0.05 +/- 0.01 nmol ATP/10(9) spermatozoa) than non-stored spermatozoa. Boar spermatozoa contained similar concentrations of ATP, whether fresh (74.2 +/- 8.1 pmol ATP/10(6) spermatozoa), stored for 1 day (77.0 +/- 8.1 pmol ATP/10(6) spermatozoa) or 5 days (81.96 +/- 8.1 pmol ATP/10(6) spermatozoa). For all three species, assay variation was low (inter-assay, 0.66-1.9% CV; intra-assay, 1.3% CV).     

A comparative study on lipid peroxidation, activities of antioxidant enzymes and viability of cattle and buffalo bull spermatozoa during storage at refrigeration temperature

A comparative study was conducted to monitor the activities of some antioxidant enzymes, lipid peroxidation and viability of cattle and buffalo bull spermatozoa during storage of semen at refrigeration temperature over a period of 72 h. Semen samples, collected from six cross bred cattle bulls (group I) and six Murrah buffalo bulls (group II), were diluted in egg-yolk-citrate and the spermatozoa were separated from seminal plasma by centrifugation at 4 degrees C in a refrigerated centrifuge. The malondialdehyde (MDA) production in group I increased from 1.17+/-0.29 at 0 h to 7.50+/-0.52 nmol/10(8)spermatozoa after 72 h of storage while in group II it increased from 1.99+/-0.26 to 8.70+/-0.10 nmol/10(8)spermatozoa in the same period. However, buffalo bull spermatozoa had a significantly higher (p<0.05) lipid peroxidation at 0 h as well as at 12, 24 and 48 h (p<0.01) periods. The activities of antioxidant enzymes viz. SOD, GPx and G6PD in both the groups showed a similar pattern of change i.e. the activities declined successively in spermatozoa and increased in the seminal plasma. However, the activities of these three enzymes remained significantly higher in the cattle bull spermatozoa than that in buffalo bull spermatozoa. Amount of MDA produced in spermatozoa of both the groups was negatively correlated while SOD, GPx and G6PD activities in spermatozoa were positively correlated to the motility and viability of spermatozoa. Sperm motility as well as viability was significantly less (p<0.05) in group II than that in group I. SOD, GPx and G6PD activities in spermatozoa of both the groups were negatively correlated to lipid peroxidation of spermatozoa cell membrane. The results showed that the less activities of antioxidant enzymes in buffalo bull spermatozoa was due to higher lipid peroxidation that indicated that they were more prone to oxidative stress as compared to cattle bull spermatozoa when stored at refrigeration temperature.     

Influence of seminal plasma on the fecundity of chicken spermatozoa

This study was undertaken to investigate the influence of seminal plasma on the fecundity of chicken sperm. Sperm diluted with either incubated seminal plasma (5 or 37 degrees C for 24 h) or seminal plasma from incubated whole semen (5 or 37 degrees C for 24 h) had lower fertility levels and motility scores than sperm diluted in either fresh seminal plasma or a synthetic diluent. The number of sperm with damaged membranes increased with seminal plasma derived from 37 degrees C incubation. The depressive effect of incubated seminal plasma on semen fertility was eliminated by microfiltering .(0.22 mum) the seminal plasma either before or after incubation. Filtration of seminal plasma was only effective in eliminating the depressive effect on sperm motility when filtering was done after incubation. Filtration of seminal plasma reduced the percentage of damaged sperm in all treatments. It can be concluded that there are factors in seminal plasma that are deleterious to the fecundity of chicken spermatozoa and they may be derived from degenerating sperm and/or various fluids, cells and debris collected with the semen during manual semen collection.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.     

Association of classical semen parameters, sperm DNA fragmentation index, lipid peroxidation and antioxidant enzymatic activity of semen in ram-lambs

The objective was to determine relationships among classical semen characteristics, sperm chromatin structure assay (SCSA), lipid peroxidation and antioxidant enzymatic activity in ram-lamb semen. Fifty-seven ram-lambs were electroejaculated, and routine semen evaluation was conducted (as part of a breeding soundness evaluation). The percentage of sperm DNA fragmentation index (%DFI) and the percentage of sperm with abnormally high DNA stainability (HDS; immature spermatozoa) were determined by SCSA using the metachromatic properties of acridine orange. Semen was centrifuged at 800 x g for 15 min to separate spermatozoa and seminal plasma and the aliquots were stored at -70 degrees C until analyzed. Lipid peroxidation, superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels in seminal plasma and spermatozoa were measured by spectrophotometric assays. The classical semen parameters were negatively related to lipid peroxidation and GPx activity in spermatozoa; motility and morphology were negatively related to %DFI (P < 0.05). Based on Kruskal-Wallis pair-wise comparison of median values among breeding soundness outcome groups, %DFI was lower in the satisfactory group compared to other groups (P < 0.05) and the lipid peroxidation and GPx activity in seminal plasma and spermatozoa were lower in satisfactory and questionable groups (P < 0.05). However, the SOD was lower in the unsatisfactory group (P < 0.05). In summary, classical semen parameters were negatively related to % DFI, lipid peroxidation and GPx activity in ram-lamb spermatozoa and seminal plasma. There were indications that SOD and GPx have crucial protective roles against the toxic effect of reactive oxygen species (ROS) in ram-lamb semen.     

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo)

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation.     

Effect of dialysis on quality characteristics of turkey semen during liquid storage

Low molecular weight substances such as zinc and peroxides are present in seminal plasma and are responsible for deleterious effects in stored semen. On the contrary, molecules larger than 50 kDa are beneficial to in-vitro storage of spermatozoa. Since the effects of different seminal plasma fractions in turkey semen are not completely known, the purpose of the study was to determine the effects of turkey semen dialysis with a 12-14 kDa cut-off on viability, hypo-osmotic membrane integrity, or sperm motility of turkey spermatozoa stored up to 48 h at 5 degrees C. Twelve pools of semen, each pool originating from four toms, were used. Each pool was divided into two aliquots, one of which was dialyzed while the other represented the control. Each semen aliquot was evaluated for sperm viability, membrane integrity and motility after 6, 24 and 48 h of in-vitro storage. Cold storage of turkey semen for 48 h significantly worsened (P<0.01) sperm viability, hypo-osmotic membrane integrity, and sperm motility index of both control and dialyzed samples. After 24 and 48 h sperm viability, membrane integrity and sperm motility index were better (P<0.01) in dialyzed semen compared to the control.     

Phospholipid fatty acid composition, vitamin E content and susceptibility to lipid peroxidation of duck spermatozoa

Recent studies on chicken semen have suggested that the lipid and fatty acid composition of spermatozoa may be important determinants of fertility. Phospholipid fatty acid composition, vitamin E content and in vitro susceptibility to lipid peroxidation of duck spermatozoa were investigated using GC-MS and HPLC based methods. The total phospholipid fraction of duck spermatozoa was characterized by high proportions of the n-6 polyunsaturated fatty acids arachidonic (20:4n-6), docosatetraenoic (22:4n-6) and docosapentaenoic (22:5n-6) acids but a substantial proportion of the n-3 fatty acid docosahexaenoic (22:6n-3) acid was also present. Palmitic (16:0) and stearic (18:0) fatty acids were the major saturates in sperm phospholipids. Among the phospholipid classes, phosphatidylserine (PS) had the highest degree of unsaturation due to very high proportions of 22:6n-3, 22:5n-6, 22:4n-6 and 20:4n-6, comprising together more than 75% of total fatty acids in this fraction. Phosphatidylethanolamine (PE) also contained high proportions of these four C(20-22) polyunsaturates, which together formed 60% of total fatty acids in this phospholipid. Spermatozoa and seminal plasma of duck semen were characterized by unexpectedly low content of vitamin E, being more than 4-fold lower than in chicken semen. In duck semen the major proportion of the vitamin E (>70%) was located in the spermatozoa. The very high proportion of 22:6n-3 in PS and PE fractions of duck sperm lipids and the comparatively low levels of vitamin E could predispose semen to lipid peroxidation. Nevertheless the in vitro susceptibilities to Fe2+-stimulated lipid peroxidation of duck and chicken spermatozoa were very similar. The results of the study suggest that increased superoxide dismutase and glutathione peroxidase activity and increased antioxidant activity of seminal plasma may compensate for the low levels of vitamin E to help protect the membranes of duck spermatozoa, which exhibit a high degree of unsaturation from oxidative stress.     

Arylsulfatases are present in seminal plasma of several domestic mammals.

Mammalian spermatozoa and seminal plasma both contain high levels of arylsulfatases (AS), enzymes that remove sulfate from sulfated glycoconjugates. In ejaculated semen of boars, 85% of AS was found in seminal plasma whereas only 13% was found in spermatozoa. A comparable distribution of AS between spermatozoa and seminal plasma was observed in other domestic mammals. The presence of AS in seminal plasma was not due to leakage from spermatozoa because sperm cells had intact acrosomes and plasma membranes after their separation from seminal plasma, and because 84% of the acrosomal marker enzyme hyaluronidase was retained in washed spermatozoa. Spermatozoa in boar semen diluted with Beltsville Thawing Solution (BTS) deteriorated faster during storage at 17 degrees C than spermatozoa stored in BTS without seminal plasma. This suggests that seminal plasma has a deleterious effect on mammalian spermatozoa. We propose that (1) sulfated glycoconjugates stabilize sperm plasma membranes; (2) AS present in seminal plasma contribute to the deterioration of spermatozoa by desulfating these glycoconjugates; and (3) AS present in seminal plasma could well play a role in sperm capacitation.     

Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation

Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.     

Effect of native phosphocaseinate on the in vitro preservation of fresh semen

The fertilization capacity of goat sperm stored in milk extenders is approximately 12-24h. Long-term storage of goat sperm (up to 3 days) is desirable as it would confer greater flexibility to breeding farms. The aim of this study was to evaluate in vitro motility parameters of buck spermatozoa for up to 7 days of storage using skim milk or chemically defined extender supplemented with native phosphocaseinate (NPPC). Four experiments were conducted to determine optimum temperature (4 or 15 degrees C) and storage conditions (aerobic versus anaerobic), the effect of seminal plasma on sperm survival, the optimal concentration of NPPC and the effect of beta lactoglobulin (BL). Both skim milk and NPPC were found to be more efficient for preserving goat sperm at 4 degrees C than at 15 degrees C (P<0.01). Furthermore, when sperm was stored at 4 degrees C, no detrimental effects of seminal plasma were observed. Our results showed that motility parameters can be maintained with success until Day 4. However, NPPC-based extenders extend the in vitro survival to 7 days of storage. The optimal concentration of NPPC for the preservation of sperm cells for 4 days of storage was 81g/l and for 7 days of storage was 81 and 54g/l. No effect of the supplementation of the NPPC extender with BL was found.     

Improving the fertilizing ability of sex sorted boar spermatozoa

The sex sorting of spermatozoa by flow cytometry induces damage, since sperm cells are highly diluted, affecting their functionality and fertilizing ability. In this work it was investigated whether the concentration of sex sorted spermatozoa by the sedimentation method, rather than centrifugation, in combination with the presence of the seminal plasma protein PSP-I/PSP-II heterodimer may improve their fertilizing ability. Spermatozoa were sorted by flow cytometry and collected in BTS with 10% of seminal plasma (group C: control) or with 1.5mg/mL of PSP-I/PSP-II heterodimer (group H). Collected spermatozoa from each medium were split into two aliquots. One aliquot of each group was centrifuged (800 x g/5 min) just after sorting and stored 16-18 h at 17 degrees C (groups Cc and Hc) at 6 x 10(6)sperm/mL. The second aliquot was directly stored at 17 degrees C for 16-18 degrees C (group Cs and Hs). After storage the supernatant was discarded and the sedimented pellet adjusted to 6 x 10(6)sperm/mL. Membrane integrity, acrosome status and motility characteristics of spermatozoa from all groups were assessed. Post-weaning pre-ovulatory sows were inseminated by laparoscopy into the oviduct with 0.3 x 10(6) sex sorted spermatozoa to assess their ability to penetrate oocytes in vivo. Putative zygotes were collected 18 h after insemination by washing the oviduct. Penetration and monospermic rates were evaluated. After 16-18 h of storage, centrifuged spermatozoa collected with 10% seminal plasma or 1.5 mg/mL PSP-I/PSP-II heterodimer after sex sorting showed lower (p<0.05) percentages of membrane integrity, motility and fertilization than sedimented spermatozoa. Overall, the presence of 10% seminal plasma or PSP-I/PSP-II heterodimer did not affect the results. However, a positive effect of PSP-I/PSP-II heterodimer (p<0.05) was observed in sedimented spermatozoa. Hence, our results indicate that the sedimentation method in the presence of PSP-I/PSP-II heterodimer improves the in vivo fertilizing ability of sex sorted boar spermatozoa.