Effect of N-acetyl-D-glucosamine, and glycerol concentration and equilibration time on acrosome morphology and motility of frozen-thawed boar sperm.

A series of experiments were conducted to determine the effect of N-acetyl-D-glucosamine, glycerol concentration and equilibration time for the freezing of boar spermatozoa in 5 ml maxi-straws. The optimum final glycerol concentration in the diluent with 0.05% N-acetyl-D-glucosamine in the first diluent was 2-3% and the optimum glycerol equilibration time was 2-3h. In conclusion, we recommend the first diluent containing 11% lactose hydrate, 20% egg yolk and 0.05% N-acetyl-D-glucosamine in 100ml distilled water, and the second diluent containing 11% lactose hydrate, 20% egg yolk, 4% glycerol and 1% orvus es paste for the diluents of boar sperm freezing. Also, we found out that 0.05% soluble N-acetyl-D-glucosamine was the optimum concentration in the first diluent and a concentration of 0.05% soluble N-acetyl-D-glucosamine significantly enhanced the cryopreservation of boar spermatozoa.     

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility.     

Duration of thawing on post thaw acrosome morphology and motility of boar spermatozoa frozen in 5-ml maxi-straws

Ejaculates from six boars were frozen in 5-ml maxi-straws and stored in liquid nitrogen. Maxi-straws were thawed by submersion in a 52 degrees C circulating waterbath for 28, 34, 40, 46, 52, or 58 sec. The post thaw percentages of motile sperm and acrosomes with a normal apical ridge (NAR) at the initial microscopic examination were significantly higher (P < 0.05) for maxi-straws submerged for 40 sec than for any other duration of submersion. The mean core temperature of maxi-straws after 40 sec of submersion was 4.8 degrees C. Based on regression equations for post thaw motility and NAR acrosomes, a 37 sec thawing time would be expected to provide the maximal post thaw survival for boar sperm frozen in maxi-straws.     

1-O-alkylglycerols improve boar sperm motility and fertility.

1-O-alkylglycerols are naturally occurring ether lipids with potent biological activities. They may interfere with lipidic signaling, and they amplify platelet-activating factor (PAF) biosynthesis in a monocyte cell line. The PAF is produced by mammalian sperm and is an important activator of sperm motility. The aim of this study was to evaluate the effect of in vitro treatment of boar spermatozoa with natural 1-O-alkylglycerols (10 microM) on 1) boar sperm motility; 2) production of PAF and its metabolite, lyso-PAF, by spermatozoa; and 3) fertility in artificial inseminations of breeding sows. Using a computer-assisted spermatozoa analyzer, we found that 1-O-alkylglycerols increased percentage motility as well as velocity parameters after 24 h. These effects were partially or totally reversed by the PAF receptor-antagonist SR 27417. After [3H]-1-O-alkylglycerol incubation with boar spermatozoa, we identified [3H]lyso-PAF by high-performance liquid chromatography. Production of PAF and lyso-PAF was measured with a biological assay using [3H]serotonin release from rabbit platelets. 1-O-alkylglycerols significantly increased lyso-PAF production but had no effect on PAF production. The effect of 1-O-alkylglycerols on fertilization was also evaluated in industrial breedings: 1-O-alkylglycerol-treated or untreated semen dilutions were alternately used for artificial inseminations of sows on 12 farms. 1-O-alkylglycerol treatment increased the number of farrows but had no effect on the mean size of the litters. This study demonstrates that 1-O-alkylglycerol treatment of boar spermatozoa in vitro improves their motility and fertility, and it suggests that this effect is related to PAF metabolism and function in boar spermatozoa.     

Lead chloride affects sperm motility and acrosome reaction in mice

Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl₂) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl₂/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte.     

Viability, acrosome morphology and fertilizing capacity of boar spermatozoa treated with strontium chloride

The positive effect of strontium ions (Sr2+) on sperm motility, capacitation and acrosome reaction has been demonstrated in the mouse, human, guinea pig and hamster. In the present study, we have evaluated the effect of Sr2+ on the viability and acrosome morphology of boar spermatozoa, and on the fertilization and development after the microinjection of Sr(2+)-treated spermatozoa into porcine oocytes. Before incubation, 79% of spermatozoa were classified as propidium iodide (PI)-negative (live) using the LIVE/DEAD Sperm Viability Kit. After incubation with strontium chloride (SrCl2), 39% (0 mM; no divalent cations), 25% (1.9 mM) and 24% (7.5 mM) of them were classified as PI-negative. The proportion of spermatozoa that had initiated the acrosome reaction was higher in Sr(2+)-containing medium than in Sr(2+)-free medium, when assessed by electron microscopy. There was no significant difference in percentage of spermatozoa initiating the acrosome reaction between Sr2+-treated groups (1.9 mM: 22%, 7.5 mM: 33%, p>0.05). After the microinjection of spermatozoa incubated with SrCl2, 67% (1.9 mM) and 61% (7.5 mM) of injected oocytes were successfully fertilized, and then 43% (1.9 mM) and 41% (7.5 mM) contained a fully decondensed sperm head. Sham-injected oocytes were significantly activated at a lower rate than Sr(2+)-treated groups (27%, p<0.05). Next, after microinjection of spermatozoa incubated with 1.9 mM SrCl2 (n=51), 45% of injected oocytes cleaved on day 2, and 18% developed to blastocysts on day 7 (sham-injection, n=48: 15% to cleavage and 0% to blastocyst). These results demonstrate that Sr2+ is likely to positively affect the fertilizing capacity of spermatozoa in the pig.     

Influence of cryoprotectants glycerol and amides, combined with antioxidants on quality of frozen-thawed boar sperm

The present study was undertaken to examine whether the cooling and freezing extenders containing a mixture of antioxidants (AOs) catalase, Na-pyruvate and mercaptoethanol and one of three types of cryoprotectants (CPs) would be able to improve the quality of frozen-thawed boar sperm. The collected semen, only the sperm-rich fraction, was diluted 1:1 with Androhep plus? extender, stored at 15°C for 2 h and centrifuged. The centrifuged sperm pellet was re-suspended in lactose-egg yolk extender and divided into four groups for mixing with freezing extenders containing different kinds of CPs at 5°C: (I) glycerol (GLY) as control; (II) GLY with AOs; (III) dimethyl formamide (DMF) with AOs and (IV) dimethyl acetamide (DMA) with AOs. Processed sperm were packaged in 0.25-mL straws and frozen using a controlled rate freezer. After thawing, the diluted thawed sperm were incubated at 38°C for 10 min and was assessed for motility by CASA, membrane/acrosome integrity by FITC-PNA/PI and DNA integrity (DFI) by SCSA. All sperm parameters evaluated, except DFI, were negatively affected (P<0.001) when using DMF (III) or DMA (IV) as CPs instead of GLY (I and II). Total sperm motility was lower (P<0.001) in the samples supplemented with AOs (32.4 ± 1.2, 23.9 ± 1.5, 6.9 ± 0.7, and 10.3 ± 0.9%, for treatments I, II, III and IV, respectively). The quality of sperm frozen in DMF was not different from DMA (P>0.05). There was no difference in DFI among the studied groups (P>0.05). In conclusion, based on the present results, addition of AOs to cooling and freezing extenders and/or replacement of GLY with DMF or DMA could not improve quality of frozen-thawed boar sperm.     

In vitro fertility and motility characteristics of frozen-thawed boar epididymal spermatozoa separated by Percoll

Frozen-thawed epididymal spermatozoa from four boars were separated through a Percoll gradient, and motility characteristics and in vitro fertility were assessed. Percoll-separated spermatozoa had a significantly higher percentage of motile and progressively motile spermatozoa than those that were not separated (P < 0.0001). However, there were no clear differences in other motility parameters between Percoll-separated and un-separated spermatozoa. Furthermore, sperm agglutination was decreased by Percoll separation (P < 0.05). The effects of Percoll separation on in vitro fertility of spermatozoa differed among boars. In addition, there were large differences in fertility between sperm samples in vitro. Sperm samples, which indicate highly motile and progressively motile, did not always show high in vitro fertility. Furthermore, there was no distinct pattern between fertility in vitro and motility parameters. There was no difference in fertility in vitro between Percoll-separated and un-separated spermatozoa from two of the four boars. However, in vitro fertility of Percoll-separated spermatozoa was higher than that of un-separated spermatozoa from the other two boars.     

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.     

Origin of a sperm motility inhibitor from boar seminal plasma

We have investigated the origin of the sperm motility inhibitor (SPMI) from boar seminal plasma. SPMI was measured by its capacity to inhibit the motility of demembranated spermatozoa and by an enzyme-linked immunosorbant assay (ELISA). Among the various reproductive and now reproductive tissues and fluids tested, only the seminal vesicle fluid and seminal plasma contained significant amounts of SPMI biological activity and SPMI antigen. Like other seminal vesicle fluid proteins, SPMI is diluted 6- to 8-fold upon ejaculation. By immunohistochemical detection at the light microscope with antibodies obtained from rabbits immunized with SPMI purified from boar seminal plasma, SPMI was found in the cytosol and/or on the plasma membrane bordering the lumen of the seminal vesicles. At the electron microscope level, SPMI appeared to be present only on the surface of the secretory cells. The data indicate that SPMI originates from a single tissue, the seminal vesicle, and suggest that only the mature form present on the luminal surface of the gland can react with the antibody generated from rabbits immunized with the secreted form of SPMI. © 1993 Wiley-Liss, Inc.     

Measurement of boar sperm motility by the trans-membrane migration method

The conventional microscopic methods for evaluating sperm motility of domestic animals are mostly inadequate due to their subjectivity and lack of precision. Recently, a trans-membrane migration method, originally developed for the examination of human sperm motility, has substantially overcome these problems. This study investigated the applicability of the method to boar sperm motility measurement. The apparatus used was simple and consisted only of syringe plungers, poriferous membranes, and modified multi-well culture plates. It measured the proportion of sperm in the semen that moved across the membrane after incubation at 37 degrees C for 3 hr. The sperm motility as measured by this method correlated well with that measured by direct microscopic examinations. The measurement was more reliable using an 8-microns instead of a 5-microns pore-size membrane. The method was found to work equally well for the sperm motility measurement of the semen with a sperm concentration between 1.5 x 10(8)/ml and 6.0 x 10(8)/ml. The results indicate that this method is a simple, objective, quantitative, and reproducible design for the measurement of boar sperm motility.     

Functional integrity of sex-sorted, frozen-thawed boar sperm and its potential for artificial insemination

Despite being developed in its present form 20 years ago, sex-sorting of mammalian sperm is still a work in progress. While relatively successful in cattle and sheep, the unique challenges of incorporating sex-sorted sperm into pig production have not yet been overcome. Generally speaking, boar sperm survive freeze-thawing less well and are required in larger numbers for insemination, while in vitro embryo production of pig embryos is less successful compared to other domestic species [Niemann H, Rath D. Progress in reproductive biotechnology in swine. Theriogenology 2001;56:1291-1304]. Due to the large number of sperm required for artificial insemination in pigs, a technique of storing sperm after sorting must be developed while adequate numbers of sperm are allocated into X- or Y-chromosome-bearing enriched pools. Cryopreservation is perhaps the ideal method of storage between sorting and insemination, as it allows unlimited time to build up a sperm bank, whereas liquid-storage requires the use of sperm within days of sorting. The limited number of studies investigating the survivability of sex-sorted, frozen-thawed boar sperm have produced promising in vitro results but poor in vivo outcomes. Before fertility can be improved, the causes of any damage to sperm function during the sex-sorting and freeze-thawing procedures must be more fully understood. Once defined, the source of damage may be minimised and this would lead to increased success rates after in vivo application of sex-sorted, frozen-thawed boar sperm.     

Sperm binding glycoprotein (SBG) produces calcium and bicarbonate dependent alteration of acrosome morphology and protein tyrosine phosphorylation on boar sperm

The oviduct is a dynamic organ which modulates gamete physiology. Two subpopulations of sperm have been described in the oviduct of sows, a majority with normal appearance in the deep furrows and a minority, centrally located, and showing damaged membranes. Sperm-oviduct interaction provides the formation of a sperm storage and allows the selection of sperm with certain qualities. Pig (Sus scrofa) oviductal sperm binding glycoprotein (SBG) binds to sperm and exposes Gal beta1-3GalNAc. This disaccharide may be recognized by boar spermadhesin AQN1, which seems to be involved in sperm interaction with the oviduct. SBG is present at the apical surface of the epithelial cells that surround the lumen of the oviduct rather than at the bottom of the crypts. These characteristics imply it could be involved in sperm interaction with this organ. In this study, we evaluate the effect of SBG over boar sperm. We show that the presence of SBG produces alterations of the acrosome morphology of sperm only when they are incubated in capacitating conditions. SBG binds to the periacrosomal region of sperm undergoing capacitation. Its presence induces an increase on the tyrosine-phosphorylation of a polypeptide of apparent molecular mass 97 kDa, as occurs with a 95 kDa protein in other mammalian sperm upon acrosomic reaction. Altogether, these results suggest that SBG might be involved in sperm selection by alteration of the acrosome of sperm that have already begun the capacitation process when they arrive to the oviduct.     

Sperm biology and control of reproduction in sturgeon: (II) sperm morphology, acrosome reaction, motility and cryopreservation

In the present review, sperm morphology, acrosome reaction, motility, short-term storage and cryopreservation are summarized and discussed in sturgeon (Chondrostei, Acipenseriformes). The elongated head of spermatozoon comprises an acrosome with 8?C12 posterolateral projections. Usually three endonuclear canals are observed in the nucleus. Proximal and distal centrioles and 3?C6 mitochondria are located in the midpiece region. The flagellum consists of an axoneme with a typical ??9?+?2?? structure of microtubules and presents a ribon-like structure due to two lateral membranous fins. Egg water, Ca²⁺ and Mg²⁺ can trigger acrosome reaction. Trypsin- and chymotrypsin-like activities are reported in sturgeon sperm. These physiological properties of sturgeon sperm are identified as serine activity with 33?kDa molecular mass and can be inhibited by their respective inhibitors. The K⁺ prevents sperm activation in seminal plasma, and hypo-osmolality or decrease of extracellular K⁺ triggers sperm activation. Extracellular Ca²⁺ is involved in flagellar beating pattern and sperm velocity. After activation, sperm motility, velocity, and flagellar beating frequency, wavelength and amplitude decrease, while number of waves and curvature increase. Sturgeon sperm can be stored for several days at 4?°C; however it is better to add K⁺ into the immobilizing medium because it prevents sperm activation during incubation. Regarding sperm cryopreservation, methanol is a better cryoprotectant than DMSO. Either short-term storage or cryopreservation of sperm generates damage to spermatozoa that lead to reduction of sperm motility performance. Some studies suggest using an activation medium containing Ca²⁺ for enhancing sperm motility performance of incubated or frozen-thawed sperm.     

Role of sperm motility and acrosome integrity in the filtration of bovine semen

In this study, the role of sperm motility and acrosome integrity in filtration of bovine semen was investigated. In Experiment 1, the treatment of semen with formaldehyde, hyperosmotic buffer, heating and direct freezing immobilized the spermatozoa completely but their acrosomal status varied significantly (P < 0.01). The immotile spermatozoa, of any kind, did not pass through the Sephadex ion-exchange column at room temperature. In Experiment 2, semen samples possessing different percentages of immobilized spermatozoa (0, 50, 75 and 100%) were filtered through the Sephadex ion-exchange column. The immotile/dead spermatozoa were removed proportionately to their number in the semen by Sephadex ion-exchange column. The type and number of immotile spermatozoa in semen had no effect (P > 0.05) on the post-filtration recovery rate of motile spermatozoa. Filtered spermatozoa exhibited higher (P < 0.01) motility (> 90%), progressive motility (> 70%) and normal acrosomes (> 95%) than non-filtered spermatozoa. In conclusion, sperm motility seems to be more important than acrosome integrity for semen filtration, and the Sephadex ion-exchange column can remove the known quantities of different kinds dead/immotile spermatozoa.     

Effect of the addition of beta-mercaptoethanol to a thawing solution supplemented with caffeine on the function of frozen-thawed boar sperm and on the fertility of sows after artificial insemination

We have reported that artificial insemination (AI) with frozen-thawed boar semen supplemented with caffeine increased the number of uterine sperm by inhibiting the migration of polymorphonuclear leukocytes (PMNs) into the uterine lumen, thereby improving the fertility of gilts and sows. The objective of the present study was to examine the effects of the addition of the antioxidant beta-mercaptoethanol (bME) and caffeine to the thawing solution on the function of frozen-thawed sperm, on the phagocytic activity of PMNs for sperm, and on the fertility of sows after AI. When frozen-thawed sperm were cultured in the presence of 25 or 50 μm bME, sperm capacitation and spontaneous acrosome reactions were inhibited (P < 0.01). There was no effect of bME on phagocytic activity of PMNs for sperm in vitro. When hormonally treated (400 IU of equine chorionic gonadotropin + 200 IU of human chorionic gonadotropin) weaned sows experienced a single intrauterine insemination with frozen-thawed sperm (25 × 10⁸ sperm per 50 ml dose) 40 h after subsequent hCG administration, pregnancy and farrowing rates were unaffected by the addition of 50 μm bME (pregnancy rate, 20 vs 21% in controls; farrowing rate, 20 vs 21%; n = 15 and 14, respectively). However, litter size tended to be higher than in the presence of 50 μm bME compared to its absence (10.0 ± 1.0 vs 5.7 ± 1.5, respectively; P < 0.07). Thus, the addition of bME to the thawing solution containing caffeine could be of benefit for improving the function of frozen-thawed sperm without influencing the phagocytic activity of PMNs for sperm. Although there were no statistically significant effects of bME on pregnancy or farrowing rates, the litter size tended to be higher in the sows subjected to a fixed-time single AI treatment with synchronized ovulation.     

Preventive effect of Desferal on sperm motility and morphology

Transition metal ions, mainly iron, are involved in the generation of highly reactive hydroxyl radicals, which are the most powerful inducers of oxidative damage to all biomolecules. The lipids in sperm membranes are highly susceptible to oxidation. Sperm lipid peroxidation (LPO) leads to decrease of motility and reduction of likelihood for sperm‐oocyte fusion. The excess radical production may affect also the spermatozoa morphology. The aim of the present study was to investigate the effect of Desferal on the LPO, motility, and morphology of boar sperm subjected to oxidative stress. After collection, the ejaculates were equally separated and diluted in a commercial semen extender (experiment 1) or in physiological saline (experiment 2). The ejaculates of the 2 experiments were divided into aliquots, which were incubated with one of the following agents: FeSO₄ (0.1mM), H₂O₂ (0.5mM), or FeSO₄ + H₂O₂ (Fenton system), in the presence or absence of Desferal. The application of Desferal in the incubation medium had a protective effect against FeSO₄ + H₂O₂‐induced sperm damage, namely, decrease of LPO; decrease the quantity of immotile spermatozoa and decrease the number of morphological abnormalities, regardless of the used medium. In experiment 2, the presence of FeSO₄ in the incubation medium induced LPO in the same range as the combination FeSO₄ + H₂O₂, in which the effect was reduced by Desferal. Thus, the supplement of Desferal to media used for sperm storage and processing could be a useful tool for diminishing oxidative injury and improving the quality of the semen.     

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pH_i) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na⁺-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pH_i is more acidic than the external medium (pH_o = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na⁺ to sperm suspended in Na⁺-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pH_i by 0.4–0.5 pH units. That this pH_i change is the causal trigger of motility was suggested by experiments using NH₄Cl and nigericin, which increased the pH_i and resulted in activation of motility in the absence of Na⁺. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pH_i was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca²⁺ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pH_i by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca²⁺ uptake. Either mitochondrial agents or the removal of external Ca²⁺ could also block this pH_i change suggesting a similar mechanism is involved.