Sperm Tail Membrane Research Articles

Use of principal component analysis to evaluate variability in CASA and flow cytometer sperm characteristics

An intact and functional sperm plasmalemma has a major role in sperm motility and fertilizing capacity. Several techniques have been developed to evaluate the integrity of the sperm plasma membrane, but there are still some inconsistencies concerning the methods that are more closely associated with sperm function. In this study, the aim was to: i) evaluate the integrity of the boar sperm plasmalemma during 72 h of semen storage at 17 °C using four techniques: eosin/nigrosin (E/N), propidium iodide/carboxyfluorescein diacetate (PI/CFDA), hypo-osmotic swelling test (HOST), and combined HOST with eosin staining (HOST/E), ii) assess the correlations and the limits of consistency among these techniques, iii) and estimate the relationships with the acrosomal status and sperm kinetics. Results indicate that the integrity of the sperm plasmalemma decreases during 72 h of storage, although significant differences were found only using the HOST and HOST/E techniques. Moreover, use of E/N and PI/CFDA results in greater values relative to the undamaged sperm membrane than use of HOST and HOST/E at any incubation time. Overall, results using all techniques were consistent and correlate except for findings with PI/CFDA and HOST, which was slightly below 95%. Moreover, values using the techniques for the evaluation of the integrity of the sperm head and tail membranes are positively associated with the acrosomal status and different kinetic variables with the tail integrity being related to rapid linear trajectories and the head integrity to rapid curvilinear trajectories. The results of this study provide new insights into the relevance of evaluating the boar sperm plasmalemma in the routine spermiogram.

Effect of serum antioxidant levels on sperm function in infertile male

Abstract Antioxidant and reactive oxygen species (ROS) has been implicated in male infertility. Antioxidants are compounds which scavenge formation or oppose of the actions ROS. Spermatozoa are particularly vulnerable to ROS because of inherent deficiencies in intracellular antioxidant enzyme protection, thus total body antioxidant capacity become more substantial to protect sperm. In this study to try to evaluate the role of serum total antioxidant capacity, uric acid, glutathione, total thiols, ferritin, albumin and malodialdehyde in male infertility. 66 men included in this study 40 were infertile and 26 were healthy fertile. Results showed a significant decrease in sperm progressive motility and a significant increase in sperm tail abnormality, also there were a significant decrease in serum total antioxidant capacity, uric acid and albumin in infertile group. Because of lack of the intracellular antioxidant enzymes sperm cannot protect plasma membrane that surrounds the tail, the protection depends on the antioxidant defense afforded by the seminal plasma and on the entire body antioxidant capacity. Decrease of serum antioxidant capacity, uric acid and albumin may cause destroy of sperm tail membrane and this may lowering of sperm progressive motility.

Integrity of head and tail plasmalemma is associated with different kinetic variables in boar sperm

An intact and functional sperm plasmalemma has a major role in sperm motility and fertilizing capacity. Several techniques have been developed to evaluate the integrity of the sperm plasma membrane, but there are still some inconsistencies concerning the methods that are more closely associated with sperm function. In this study, the aim was to: i) evaluate the integrity of the boar sperm plasmalemma during 72h of semen storage at 17°C using four techniques: eosin/nigrosin (E/N), propidium iodide/carboxyfluorescein diacetate (PI/CFDA), hypo-osmotic swelling test (HOST), and combined HOST with eosin staining (HOST/E), ii) assess the correlations and the limits of consistency among these techniques, iii) and estimate the relationships with the acrosomal status and sperm kinetics. Results indicate that the integrity of the sperm plasmalemma decreases during 72h of storage, although significant differences were found only using the HOST and HOST/E techniques. Moreover, use of E/N and PI/CFDA results in greater values relative to the undamaged sperm membrane than use of HOST and HOST/E at any incubation time. Overall, results using all techniques were consistent and correlate except for findings with PI/CFDA and HOST, which was slightly below 95%. Moreover, values using the techniques for the evaluation of the integrity of the sperm head and tail membranes are positively associated with the acrosomal status and different kinetic variables with the tail integrity being related to rapid linear trajectories and the head integrity to rapid curvilinear trajectories. The results of this study provide new insights into the relevance of evaluating the boar sperm plasmalemma in the routine spermiogram.

Mitigation of sperm tail abnormalities using demembranation approach in the clouded leopard (Neofelis nebulosa).

Clouded leopards (Neofelis nebulosa) produced high proportion of abnormal spermatozoa (mainly tail defects) that can limit sperm movement and conception. The study aimed to better identify the origin of those defects using a demembranation approach. Ejaculates (1-2 ejaculations/male; n=9) were allocated to simple washing (control; resulting in 11.7%±1.9% coiled tails) and processed through colloid centrifugation to reduce the number of sperm with tail defects (treatment, resulting in 5.9%±0.9% coiled tails). Aliquots of semen were incubated in hypo-osmotic solution (HOS, 60 mOsm fructose solution) containing 5mM dithiothreitol (DTT) (a reducing agent) to prevent oxidation of sperm membrane. Thereafter, 20% Triton X-100 (TX) (a detergent) was added to the HOS/DTT-treated samples. After HOS/DTT incubation, the control samples and sperm-selected samples presented 73.4%±3.1% and 73.9%±2.5% swollen sperm (bent and coiled) indicating membrane intact, respectively. Most of the coiled tail in the raw ejaculates could not be opened by TX indicating that the cause of coiled sperm tails may be from testicular origin. The proportion of sperm with tightly coiled tail tended to be lower in the sperm-selected group than control group (18.8%±3.8% and 26.5%±3.4%; p=.1), whereas the sperm opened up by TX tended to be higher in the sperm-selected group (53.6%±10.4% and 21.1%±7.9%; p=.06). The results indicated TX was able to uncoil half of the tightly coiled sperm in the semen undergone preparation. In conclusion, the coiled sperm in the clouded leopard semen were likely not a defect of sperm volume regulation during post-ejaculate (osmotic swelling) but pre-ejaculate origin. Semen preparation demonstrated its ability to lessen the primary sperm defects and selected spermatozoa that were prone to be mitigated after demembranation.

Effect of copper exposure on reproductive ability in the bank vole (Myodes glareolus).

The amount of copper in natural ecosystems is steadily increasing, due to human activities. It accumulates in plants, posing a threat to herbivores. In polluted areas the population density of small rodents is observed to be lower. The decline in rodent numbers may be caused by increased mortality or diminished fertility. This study examined the effect of copper on the reproductive activity of the bank vole (Myodes glareolus), a small rodent which during foraging often wanders into fields where it might be exposed to pollution. The animals were treated with solutions of 0, 150 or 600 ppm Cu. After 12 weeks of exposure the quality and quantity of the male's sperm was tested. To assess morphological development we compared the experimental groups for body weight, the weight of the male's testes and accessory sex glands, the female's uterus, and the number of matured ovary follicles in tested females. At both doses, copper administration led to lower sperm count and caused sperm head anomalies. The higher dose compromised sperm tail membrane integrity, viability and motility. No effect of copper on morphological development was observed in males, and only the lower dose increased testes weight. In females the higher dose had a negative effect on morphological development, and the lower dose increased uterus weight. No effect of copper on ovarian follicle number was found. For the first time, the morphology of the most typical ovarian follicles of the bank vole is presented.

Sexual maturation in common vole (Microtus arvalis) males raised under laboratory conditions.

The common vole is one ofthe most numerous rodents in Europe and Asia but its reproductive biology is not fully described. It is thought that females reach reproductive abilities at a very young age, however, there is no data concerning male sexual maturation. The aim of the present study was to determine the rate of sexual maturation of males of the common vole. Research was carried out on 4, 6, 8 and 10 week-old animals. Body, testes and accessory sex glands weights were compared to evaluate morphological development. Epididymal sperm quality was assessed by a motility test, sperm tail membrane integrity, viability, maturity and sperm head morphology. Moreover, the number of sperm cells in 1 mm3 of semen was evaluated. The largest body weights were observed in 8 and 10 week-old males, which also possessed the highest relative weights of gonads and accessory sex glands. These groups of males produce about 2 times more gametes than 6 week-old individuals. The highest seamen quality was noted in 8 and 10 week-old males. Based on the obtained results, it is concluded that among the investigated age groups the most appropriate male age for fertile copulation is between 8 and 10 weeks.

Semen quality parameters and embryo lethality in mice deficient for Trp53 protein

Trp53 is a protein which is able to control semen parameters in mice, but the extent of that control depends on the genetic background of the mouse strain. Males from C57BL/6Kw, 129/Sv, C57BL×129 -p53+/+ (wild type controls) and C57BL×129-p53-/- (mutants) strains were used in the study, and histology and light microscopy were applied to evaluate the influence of genetic background and Trp53 (p53) genotype on testes morphology and semen quality in male mice. We showed that sperm head morphology, maturity and tail membrane integrity were controlled only by the genetic background of C57BL/6Kw and 129/Sv males, while testes weight and sperm concentration depended on both the genetic background and p53 genotype. Cell accumulation in seminiferous tubules may be responsible for heavier testes of p53-deficient males. In addition, to examine the effect of sex and p53 genotype on embryo lethality, pairs of control (C57BL×129-p53+/+) and heterozygous (C57BL×129-p53+/-) mice were examined. Before day 7 post coitum (dpc), female and male embryos were equally resorbed in both crosses types. After 7 dpc, preferential female embryo lethality in the heterozygote pairs was responsible for the skewed sex ratio in their progeny. Also, mutant female and male newborns were underrepresented in the litters of the heterozygous breeding pairs.

Aquaporins in sperm osmoadaptation: an emerging role for volume regulation

Upon ejaculation, mammalian sperm experience a natural osmotic decrease during male to female reproductive tract transition. This hypo-osmotic exposure not only activates sperm motility, but also poses potential harm to sperm structure and function by inducing unwanted cell swelling. In this physiological context, regulatory volume decrease (RVD) is the major mechanism that protects cells from detrimental swelling, and is essential to sperm survival and normal function. Aquaporins are selective water channels that enable rapid water transport across cell membranes. Aquaporins have been implicated in sperm osmoregulation. Recent discoveries show that Aquaporin-3 (AQP3), a water channel protein, is localized in sperm tail membranes and that AQP3 mutant sperm show defects in volume regulation and excessive cell swelling upon physiological hypotonic stress in the female reproductive tract, thereby highlighting the importance of AQP3 in the postcopulatory sperm RVD process. In this paper, we discuss current knowledge, remaining questions and hypotheses about the function and mechanismic basis of aquaporins for volume regulation in sperm and other cell types.

Effects of flaxseed dietary supplementation on sperm quality and on lipid composition of sperm subfractions and prostatic granules in rabbit

Lipids are the main structural/functional components of the sperm, and their composition may undergo a series of modifications in relation to either physiologic events (capacitation and acrosome reaction) and/or diet. The goals of the current study were (1) to investigate whether a flaxseed (FS) dietary supplementation could affect the lipid and fatty acid profile of sperm subfractions and of prostatic granules (PGs) and (2) to evaluate the effects of dietary FS on rabbit buck semen quality. Accordingly, 20 adult New Zealand White rabbits were fed ad libitum a control diet (CO) or a diet supplemented with 5% extruded FS. Integration of diet with FS, as a consequence of the linolenic acid (C18:3n-3; LNA; 56%), increased the dietary n-3/n-6 ratio and resulted in a substantial rearrangement of sperm fatty acid composition at the subcellular level, mainly of polyunsaturated fatty acid (PUFA)n-3 (8.3% vs. 14.3%, P < 0.05). The lipid and fatty acid profiles of sperm tail membrane were the most affected, undergoing the following significant changes: (1) a reduction by half of linoleic acid (C18:2n-6; LA) and docosapentaenoic acid (22:5n-6; DPA), and a reduction of cholesterol (−70%); (2) a concomitant increase of LNA (+65%), docosahexaenoic acid (22:6n-3; DHA; +83%), and of oleic acid (C18:1n-9, +61%). As a consequence, the sperm of FS-fed rabbits had a twice higher n-3/n-6 ratio and phospholipid/cholesterol ratio compared with the control sperm. These changes might have been on the basis of the higher responsiveness to hypo-osmotic solution and, hence, the higher sperm track speed observed for the FS group. Also, the membrane integrity and viability of the LNA-enriched sperm were both improved. On the other hand, the presence of lignans in FS might have accounted for the reduction of sperm cholesterol in the semen of FS-treated rabbits. The responsiveness of sperm to acrosome reaction was not affected by the dietary treatment probably due to supranutritional level of vitamin E and to the higher number of PGs, which are known to play a key role in sperm capacitation. In conclusion, our data showed for the first time that the integration of FS into the rabbit diet may improve sperm quality by modifying the sperm lipid composition and that the sperm subfractions and the PGs respond differently to the FS-induced lipid manipulation.

Semen quantity and quality correlate with bank vole males’ social status

Laboratory studies reveal that in several rodent species the females prefer dominant males as mating partners. Here we investigate the correlation between bank vole males' social rank and their sperm quality and quantity. We used agonistic encounters to determine males' social status. Sperm quality was assessed by its motility, viability, maturity, morphology and sperm tail membrane integrity. Relatively more dominant males were heavier than males of lower social status. The males' social position affected the testes, seminal vesicles and coagulation gland development. The weights of these reproductive organs were significantly higher in more dominant males than in more subordinate males. Sperm counts and the values of the other parameters describing sperm quality were higher in high-ranking males than in subordinates. Our results suggest that bank vole females benefit from choosing and mating with high-ranking males by obtaining more and better-quality sperm.

Gene mapping of sperm quality parameters in recombinant inbred strains of mice

The aim of this study was to map chromosomal regions containing hypothetical genes responsible for the following parameters of mouse semen quality: (1) the percentage of sperm with abnormal head morphology, (2) the level of dead spermatozoa, (3) the percentage of sperm tails with residual cytoplasmic droplets, and (4) the percentage of sperm with impaired sperm tail membrane integrity. We also analyzed any possible correlations between these parameters. The most appropriate animal model for mapping genes controlling quantitative traits (QTL, quantitative trait locus) is a set of recombinant inbred (RI) strains. The set of RI strains used in this study was derived from crosses between two inbred mouse strains, KE and CBA/Kw, which differ significantly in fertility parameters and gamete quality. We analyzed the four parameters of sperm quality in male mice from two parental strains and from 12 RI strains. The strain distribution pattern (SDP) of 187 polymorphic microsatellite markers was prepared for 20 chromosomes of the mouse genome in 12 RI strains. We correlated the SDP of these markers with the values of sperm quality parameters, using MapManager QTX software (ver. b18). The mapping procedure indicated that the percentage of sperm with abnormal head morphology is controlled by gene(s) located in chromosomal regions 11q24, 11q31 and 6q15.6. There was also a strong correlation between male body weight and the hypothetical gene(s) in chromosomal region 18q47. A detailed analysis of the genes located in these regions enabled us to prepare a list of candidate genes. We discuss the basis of the correlation between the measured parameters.

Localization and regulation of plasma membrane Ca 2+-ATPase in bovine spermatozoa

Calcium (Ca 2+) signals, produced by the opening of plasma membrane entry channels, regulate a number of functions in spermatozoa such as capacitation and motility. The mechanisms of Ca 2+ removal from the sperm, required to restore resting [Ca 2+] i , include plasma membrane Ca 2+-dependent ATPase (PMCA) isoenzymes as well as a plasma membrane Na + Ca 2+ exchanger. We have recently shown that bovine sperm PMCA is stimulated by PDC-109, a secretory protein of bovine seminal vesicles. To demonstrate the subcellular localization and regulation of bovine sperm PMCA, we have performed cell fractionation, enzyme activity determination and Western blotting studies of PMCA in spermatozoa removed from the cauda epididymidis of bull. Fractionation of sperm heads and tails resulted in a distinct association of ATPase activity with the tail membrane fraction. In vitro stimulation studies with PDC-109 using intact and fractionated sperm showed an increase in enzyme activity up to 105% in sperm tail membranes. Furthermore, thapsigargin inhibition did not alter the stimulatory effect of PDC-109 on ATPase activity, indicating that no sarco/endoplasmic reticulum Ca 2+-ATPase (SERCA), but only PMCA isoenzymes are involved in this effect. Western blotting studies using a polyvalent PMCA antibody showed the exclusive presence of a 135 kDa band in the tail plasma membrane fraction. To elucidate whether or not the stimulatory effect was a direct one or indirectly mediated through PKA and PKC activation, PKA and PKC inhibitors, respectively, were used in the Ca 2+-ATPase activity assays, which was followed by PDC-109 stimulation. The stimulatory effect of PDC-109 on PMCA was still observed under these conditions, while no phosphotyrosine proteins could be detected by Western blotting in sperm extracts following PDC-109 treatment. Co-immunoprecipitation studies, PDC-109 affinity chromatography as well as overlay blots failed to show a strong association of both PMCA and PDC-109, pointing to an indirect, perhaps phospholipid-mediated effect.

128 IMPROVED POST-THAW MOTILITY, VIABILITY, AND FERTILITY ARE ACHIEVED BY HYDROSTATIC PRESSURE-TREATED BULL SEMEN

Previously, we reported that high hydrostatic pressure (HHP) significantly improves post-thaw survival of frozen mouse and IVP bovine blastocysts, presumably from the induction of shock proteins (Pribenszky et al. 2005 Anim. Repr. Sci. 87, 143–150; 2005 Repr. Dom. Anim. 40, 338). We also have reported increased post-thaw survival of HHP-treated boar and bull semen (Pribenszky et al. 2004 Repr. Fert. Dev. 17, 199–200; 2005 Repr. Fert. Dev. 18, 162–163). We now report further data on the effect of HHP treatment on motility, viability, and fertility of frozen–thawed bull semen. HHP treatments were executed by a computer-controlled pressurizing device (Cryo-Innovation, Ltd., Budapest, Hungary). Semen of 21 bulls was diluted individually to a sperm concentration of 8 � 107 mL-1 with AndroMed� or Triladyl� extenders (MiniT�b, Tiefenbach, Germany). Diluted sperm was loaded into 0.25-mL straws at 25�C, and then divided into 2 groups: one group was treated with a HHP pulse defined and optimized earlier (30 MPa for 90 min); the other group was held at 5�C for the corresponding time. After HHP, treated samples also were placed at 5�C for 3–4 h. After equilibration at 5�C, samples were frozen (10 min at -110�C, and then plunged into LN2). Straws were thawed in a 35�C water bath for 30 s. Progressive motility was assessed by the CASA system (MiniT�b). Experiments were replicated twice for each bull. Paired t-test was used to analyze data. HHP treatment significantly increased the post-thaw motility of the frozen semen of the bull population examined. The mean of the differences was 21.14% (95% confidence interval: 13.56–28.72); P = 1.08 � 10-5. The post-thaw motility of 3, 7, and 5 of the 21 bulls increased by an additional 35–60%, 25–35%, and 10–25%, respectively; no effect was seen for 6 of the bulls. Four of the 7 bulls with low (3–19%) post-thaw motility were improved to the range of 43–70% by HHP treatment. Semen of 10 bulls was the subject of viability analysis individually. Sperm head, tail, and acrosome membrane integrity were evaluated with Kovacs-Foote staining (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) and counting 300 sperm/sample. Paired t-test was used to analyze data. With HHP treatment, the proportion of the cells with intact tail, head, and acrosome increased significantly (P d 0.008). Eighty-two cows were inseminated with HHP-treated frozen–thawed semen; the 60 days non-return rate was 90.24%, whereas the 60 days non-return rate in the same population and time without treatment was 82.3% (n = 4789). Data were analyzed with the exact binomial test. HHP treatment significantly improved the non-return rate (P = 0.035; 95% confidence interval: 0.83–1.00). HHP treatment substantially increases the post-thaw semen quality of the bull population. Also, the semen of a proportion of bulls with very low semen freezability can be increased to the range where it can be frozen commercially. Further investigations are needed, including large-scale field trials incorporating the insemination of the otherwise low freezers and the biological background of the process. This work was supported by GVOP-TST050157 and Besamungsanstalt Klessheim.

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2–4 h Giemsa staining at 25–40° C is recommended for stallion semen.

Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor

Changes in membrane potential affect ion channels and transporters, which then alter intracellular chemical conditions. Other signalling pathways coupled to membrane potential have been suggested but their underlying mechanisms are unknown. Here we describe a novel protein from the ascidian Ciona intestinalis that has a transmembrane voltage-sensing domain homologous to the S1-S4 segments of voltage-gated channels and a cytoplasmic domain similar to phosphatase and tensin homologue. This protein, named C. intestinalis voltage-sensor-containing phosphatase (Ci-VSP), displays channel-like 'gating' currents and directly translates changes in membrane potential into the turnover of phosphoinositides. The activity of the phosphoinositide phosphatase in Ci-VSP is tuned within a physiological range of membrane potential. Immunocytochemical studies show that Ci-VSP is expressed in Ciona sperm tail membranes, indicating a possible role in sperm function or morphology. Our data demonstrate that voltage sensing can function beyond channel proteins and thus more ubiquitously than previously realized.

319 USE OF PENTOXIFYLLINE AND HYALURONIC ACID FOR STALLION SPERM SEPARATION

In this study, we reduced the volume of separating media and the time of centrifugation to increase the yield of viable sperm for ICSI when few sperm are available. Hyaluronic acid (HA) was used successfully in combination with swim-up for separating bull spermatozoa (Shamsuddin and Rodriguez-Martinez 1994 Anim. Reprod. Sci. 36, 61–75). The purpose of this study was to compare the effectiveness of mini-Percoll and swim-up for low numbers of sperm treated or non-treated with HA or pentoxifylline (PX), a molecule that stimulates sperm motility (Gradil and Ball 2000 Theriogenology 54, 1041–1047). Poor-to-medium quality frozen semen (0.5-mL straws, 200 million cells/mL) from 3 stallions was used 3 times each. Two straws were thawed and mixed. Aliquots of 100 μL semen were allocated to 7 treatments. Three aliquots were incubated at 38°C for 20 min before Percoll-centrifugation (P-NT: non-treated; P-PX: 3.5 mM PX; or P-HA: 1 mg/mL HA, each in 0.25 mL chemically defined handling medium (HCDM)); one aliquot was centrifuged through Percoll without incubation (P-CON: control). Our mini-Percoll was a two-layer gradient (0.4 mL 90% and 0.5 mL 45% Percoll) in a 1.5-mL microcentrifuge tube that was centrifuged at 600g for 5 min. Then a 30-μL pellet was washed in HCDM at 300g for 5 min. At the same time, 3 aliquots of 100 μL sperm were placed in 1 mL HCDM for swim-up (S-NT: non-treated; S-PX: 3.5 mM PX; or S-HA: 1 mg/mL HA) for 30 min. A 0.65-mL aliquot of supernatant was centrifuged in HCDM at 300g for 5 min. In every treatment, the final 30-μL pellet aspirated from the bottom was evaluated. Recovery rate was determined using a hemacytometer. Sperm head, tail and acrosome membrane integrity were evaluated with Kovacs-Foote staining (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) counting 300 sperm/sample. Cells were classified into 5 categories: intact head, tail and acrosome membrane (Intact); intact head, tail, damaged acrosome (IHITDA); intact head, damaged tail; damaged head, intact tail; damaged head, tail, acrosome. P-CON and P-PX resulted in more intact sperm compared to all swim-ups. IHITDA was nonsignificantly (P &gt; 0.05) higher in P-PX than in P-CON (17 vs. 11%). Better recovery rates were found in P-CON, P-PX, and S-HA than in P-NT, P-HA, S-NT and S-PX. Hyaluronic acid increased recovery rate during swim-up, but not viability in any of the treatments. Pentoxifylline is beneficial if Percoll separation is delayed, but clarification of its effect on acrosome exocytosis is needed. Table 1. Intact and recovery rates (LS means ± SE)

Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen

Cryopreservation imposes irreversible damage to sperm membranes, such as swelling and disruption of plasma and acrosome membranes, changes in membrane fluidity, altered influx of calcium, and changes in enzyme activity. Morphological integrity of the sperm plasma membrane has been widely studied using different techniques, including exposure of spermatozoa to hypoosmotic solutions (provides information concerning the biochemical activity of the sperm tail membrane), supravital test using eosin stain (yields information regarding sperm head membrane integrity), and Trypan-blue Giemsa stain (TBG; reveals both sperm plasma membrane and acrosome integrity). The objective of this study was to combine these tests in order to provide information about the integrity of the whole sperm surface, as well as acrosome status, and determine if the results of these tests were associated with sperm in vitro fertilizing ability. Stepwise regression analyses yielded a model in which fertility (maintain variable) was expressed as a combination of the results of different spermatological parameters (independent variables). The results of a test combining supravital eosin staining of samples previously submitted to hypoosmotic swelling test (STHOS) accounted for the greatest proportion of variation in fertilization rates (78%). Inclusion of the results of dual staining with TBG increased the proportion of variation in fertility rate that could be accounted for to 82%. Therefore, sperm plasma membrane integrity and function, and acrosome integrity can be considered important variables for normal sperm function and STHOST and TBG could be used for the prognosis of the potential fertility of bovine semen samples used for IVF or AI.

The use of HOS test to evaluate membrane functionality of boar sperm capacitated in vitro.

The functional and structural integrity of sperm membrane are crucial for the viability of spermatozoa. The commonly used staining test (eosin + nigrosin) for assessing sperm membrane measures only its structural integrity. The hypoosmotic swelling test (HOS) originally developed for human sperm (Jeyendran et al. 1984) has been also applied to several species of domestic animals (bull, pig, horse, dog). The test enables to evaluate the functional status of the sperm membrane. The principle of HOS is based on water transport across the sperm tail membrane under hypoosmotic conditions. It has previously been used to assess the semen quality (Revell and Mrode 1994), to analyse fertilizing capacity (Rota et al. 2000; Perez-Llano et al. 2001) and also to detect viable, immotile cells for ICSI (Intra-cytoplasmic sperm injection) in human (Zeyneloglu et al. 2000). There are two procedures commonly used for sperm capacitation in the pig-sperm washing and incubation before insemination (Nagai 1994). Capacitation involves several changes like removing molecules coating the sperm head membrane, changes in membrane fluidity and intracellular ion concentration (Green and Watson 2001). Thus the membrane integrity as well as functionality may be affected as shown by Harrison (1996). The aim of the present study was to analyse changes in sperm membrane integrity after in vitro capacitation by use of the HOS test.

Laser-induced immobilization and plasma membrane permeabilization in human spermatozoa.

We evaluated the potential use of a non-contact, 1.48 microm wavelength diode laser for immobilization of human spermatozoa and permeabilization of the sperm membrane in different culture media. When we applied a single laser shot near to the middle region of the sperm tail, spermatozoa could be immobilized either temporarily or permanently, depending on the energy used. Above an energy of 2 mJ in polyvinylpyrrolidone and 2-3 mJ in culture medium, a reliable permanent immobilization was achieved by permeabilization of the sperm tail membrane. We then explored the use of a double laser shot technique. Spermatozoa were temporarily immobilized by a first laser shot applied near to the sperm tail followed by permeabilization with a second laser shot aimed directly at the sperm tail. This sequential approach yielded permanent immobilization at much lower energy values compared with the single shot technique. Following the injection of laser-treated spermatozoa, mouse oocytes underwent normal activation and pronuclear formation. We conclude that a non-contact 1.48 microm diode laser system can be used for immobilization of spermatozoa and for permeabilization of the sperm tail membrane. This laser procedure may offer an alternative to currently used sperm pretreatment prior to intracytoplasmic sperm injection.

Evaluation of sperm tail membrane integrity by light microscopy

During routine evaluation of trypan blue-Giemsa stained semen smears, sperm cells can be found with unstained heads and with stained tails. It was hypothesized that these cells were immotile and should not be considered as live. Sperm motility was determined in isoosmotic, and presumably isotonic trypan blue-stained wet preparations. Bull, ram and boar semen smears were stained with hypoosmotic trypan blue-Giemsa to compare the relationship between the percentage of stained sperm tails and the percentage of sperm tails remaining straight under hypoosmotic conditions. Actively moving spermatozoa with unstained heads, but with stained tails were never observed in wet preparations. The correlation coefficient found between the percentage of sperm with stained tails and the percentage with straight tails was 0.81, 0.94 and 0.85 for bull, ram and boar spermatozoa, respectively. Results of this study show that sperm cells with an intact head membrane, but a stained and presumably membrane-damaged tail are not motile. Therefore these cells should be included in the dead category rather than alive in the usual live-dead studies with vital stains.