Characterization of Ras k 1 a novel major allergen in Indian mackerel and identification of parvalbumin as the major fish allergen in 33 Asia-Pacific fish species.

BACKGROUND: Fish is a well-recognized cause of food allergy and anaphylaxis. The evolutionary and taxonomic diversity of the various consumed fish species pose a challenge in the identification and characterization of the major fish allergens critical for reliable diagnostics. Globally, fish is a rising cause of food allergy complicated by a large under-investigated variety of species as well as increasing global tourism and trade. This is the first comprehensive study on allergen profiles of heat-processed fish from Vietnam. OBJECTIVE: The aim of this study was to identify the major heat-stable allergens from frequently exported Asia-Pacific freshwater and marine fish and to characterize the major allergen parvalbumin (PV) from one of the most consumed and exported fish species from Asia, the Indian mackerel (Rastrelliger kanagurta). METHODS: Heated protein extracts from 33 fish species were separated by gel electrophoresis. PV isoforms were identified by immunoblotting utilizing 3 different PV-specific monoclonal and polyclonal antibodies and further characterized by mass spectrometry. IgE reactivity was investigated using sera from 21 patients with confirmed fish allergy. RESULTS: Heat-stable IgE-reactive PVs, with up to 5 isoforms per species, were identified in all 33 analysed fish species. In the Indian mackerel, 7 PV isoforms were identified by 2D-gel electrophoresis combined with mass spectrometric analyses. The amino acid sequence deduced from cDNA of the most expressed isoform showed a high identity (>90%) to PVs from 2 other mackerel species. CONCLUSIONS AND CLINICAL RELEVANCE: Different PVs were identified as the major heat-stable allergens in all 33 analysed freshwater and marine fish species from Vietnam, many of which are exported world-wide and 21 species that have never been investigated before. The Indian mackerel PV represents a novel fish allergen, now officially registered as Ras k 1. Improved diagnostics for fish allergy against Asia-Pacific species should be developed with focus on PV.

Allergenicity of bony and cartilaginous fish - molecular and immunological properties.

Allergy to bony fish is common and probably increasing world-wide. The major heat-stable pan-fish allergen, parvalbumin (PV), has been identified and characterized for numerous fish species. In contrast, there are very few reports of allergic reactions to cartilaginous fish despite widespread consumption. The molecular basis for this seemingly low clinical cross-reactivity between these two fish groups has not been elucidated. PV consists of two distinct protein lineages, α and ß. The α-lineage of this protein is predominant in muscle tissue of cartilaginous fish (Chondrichthyes), while ß-PV is abundant in muscle tissue of bony fish (Osteichthyes). The low incidence of allergic reactions to ingested rays and sharks is likely due to the lack of molecular similarity, resulting in reduced immunological cross-reactivity between the two PV lineages. Structurally and physiologically, both protein lineages are very similar; however, the amino acid homology is very low with 47-54%. Furthermore, PV from ancient fish species such as the coelacanth demonstrates 62% sequence homology to leopard shark α-PV and 70% to carp ß-PV. This indicates the extent of conservation of the PV isoforms lineages across millennia. This review highlights prevalence data on fish allergy and sensitization to fish, and details the molecular diversity of the two protein lineages of the major fish allergen PV among different fish groups, emphasizing the immunological and clinical differences in allergenicity.

EAACI Molecular Allergology User's Guide.

The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

Shellfish allergy.

Seafood plays an important role in human nutrition and health. The growing international trade in seafood species and products has added to the popularity and frequency of consumption of a variety of seafood products across many countries. This increased production and consumption of seafood has been accompanied by more frequent reports of adverse health problems among consumers as well as processors of seafood. Adverse reactions to seafood are often generated by contaminants but can also be mediated by the immune system and cause allergies. These reactions can result from exposure to the seafood itself or various non-seafood components in the product. Non-immunological reactions to seafood can be triggered by contaminants such as parasites, bacteria, viruses, marine toxins and biogenic amines. Ingredients added during processing and canning of seafood can also cause adverse reactions. Importantly all these substances are able to trigger symptoms which are similar to true allergic reactions, which are mediated by antibodies produced by the immune system against specific allergens. Allergic reactions to 'shellfish', which comprises the groups of crustaceans and molluscs, can generate clinical symptoms ranging from mild urticaria and oral allergy syndrome to life-threatening anaphylactic reactions. The prevalence of crustacean allergy seems to vary largely between geographical locations, most probably as a result of the availability of seafood. The major shellfish allergen is tropomyosin, although other allergens may play an important part in allergenicity such as arginine kinase and myosin light chain. Current observations regard tropomyosin to be the major allergen responsible for molecular and clinical cross-reactivity between crustaceans and molluscs, but also to other inhaled invertebrates such as house dust mites and insects. Future research on the molecular structure of tropomyosins with a focus on the immunological and particularly clinical cross-reactivity will improve diagnosis and management of this potentially life-threatening allergy and is essential for future immunotherapy.

Determinants of asthma phenotypes in supermarket bakery workers.

While baker's asthma has been well described, various asthma phenotypes in bakery workers have yet to be characterised. Our study aims to describe the asthma phenotypes in supermarket bakery workers in relation to host risk factors and self-reported exposure to flour dust. A cross-sectional study of 517 supermarket bakery workers in 31 bakeries used a questionnaire, skin prick tests, and specific immunoglobulin E to wheat, rye and fungal alpha-amylase and methacholine challenge testing. The prevalence of probable occupational asthma (OA, 13%) was higher than atopic (6%), nonatopic (6%) and work-aggravated asthma (WAA, 3%) phenotypes. Previous episodes of high exposure to dusts, fumes and vapours causing asthma symptoms were more strongly associated with WAA (OR 5.8, 95% CI 1.7-19.2) than OA (2.8, 1.4-5.5). Work-related ocular-nasal symptoms were significantly associated with WAA (4.3, 1.3-13.8) and OA (3.1, 1.8-5.5). Bakers with OA had an increased odds ratio of reporting adverse reactions to ingested grain products (6.4, 2.0-19.8). OA is the most common phenotype among supermarket bakery workers. Analysis of risk factors contributes to defining clinical phenotypes, which will guide ongoing medical surveillance and clinical management of bakery workers.

Differential requirements for interleukin (IL)-4 and IL-13 in protein contact dermatitis induced by Anisakis.

BACKGROUND: Exposure to antigens of the fish parasite Anisakis is associated with the development of protein contact dermatitis in seafood-processing workers. Understanding the basic mechanisms controlling allergic sensitization through the skin is critical for designing therapies that will prevent the progression of allergic disease. OBJECTIVE: To investigate the roles of interleukin (IL)-4, IL-13 and the IL-4Ralpha in both local skin pathology and systemic sensitization following epicutaneous exposure to Anisakis proteins. METHODS: BALB/c wild-type (WT) mice and mice deficient in IL-4, IL-13 or IL-4 and IL-13, as well as mice with cell-specific impairment of IL-4Ralpha expression, were sensitized to Anisakis antigen by repeated epicutaneous application of Anisakis extract. Following this sensitization, skin pathology was recorded and systemic responses were investigated. Intravenous challenge with Anisakis extract was performed to test for the development of biologically relevant systemic sensitization. RESULTS: In WT mice, epicutaneous sensitization with Anisakis larval antigens induced localized inflammation, epidermal hyperplasia, production of T(H)2 cytokines, antigen-specific IgE and IgG1. Intravenous challenge of sensitized mice resulted in anaphylactic shock. Interestingly, IL-13 deficient mice failed to develop epidermal hyperplasia and inflammation, whilst anaphylaxis was reduced only in strains deficient either in IL-4 only, or deficient in IL-4 and IL-13 concurrently, as well as in mice deficient in IL-4Ralpha or with impaired IL-4Ralpha expression on CD4(+) T cells. CONCLUSIONS: Interleukin-13 plays a central role in protein contact dermatitis associated with repeated epicutaneous exposure to Anisakis extract, whereas IL-4 drives systemic sensitization and resultant anaphylactic shock.

Risk factors for allergy due to the two-spotted spider mite (Tetranychus urticae) among table grape farm workers.

BACKGROUND: Recent studies have reported an increased prevalence of respiratory symptoms among farm workers. The aim of this study was to identify risk factors associated with spider mite allergy among table grape farm workers. METHODS: A cross-sectional study of 207 workers in table grape farms was conducted. Skin prick tests used extracts of 8 common aeroallergens, grape mould (Botrytis cinerea) and Tetranychus urticae. Specific IgE to T. urticae was quantified using ImmunoCAP (Phadia). Erythrocyte cholinesterase levels were determined using the Test-mate ChE Cholinesterase Test System (model 460). RESULTS: The average duration of employment of workers was 10 years and 12% were pesticide crop sprayers. Work-related wheeze (26%), ocular-nasal (24%) and urticaria/skin symptoms (14%) were more prevalent in the orchards. The prevalence of sensitization (skin prick test) was highest to T. urticae (22%) followed by house dust mite (16%), with 25% being atopic. The prevalence of allergy to T. urticae (skin reactivity and work-related symptoms) was 9.5%, with respiratory allergy (6%) more common than skin allergy (3%). Work-related ocular-nasal (OR = 4.9) and skin (OR = 3.7) symptoms were more commonly reported by pesticide crop sprayers. Workers with T. urticae-allergic rhino-conjunctivitis and probable asthma were more likely to be atopic, spray pesticides and have low (<30 U/g hemoglobin) erythrocyte cholinesterase levels. CONCLUSIONS: This study demonstrated that spider mite, T. urticae, is an important outdoor allergen among table grape farm workers. The increased risk of spider mite allergy appears to be related to high pesticide exposure among crop sprayers.

Immunohistochemical localization of CYP1A, vitellogenin and Zona radiata proteins in the liver of swordfish (Xiphias gladius L.) taken from the Mediterranean Sea, South Atlantic, South Western Indian and Central North Pacific Oceans.

Cytochrome P4501A (CYP1A) monoxygenase, vitellogenin (Vtg) and Zona radiata proteins (Zrp) are frequently used as biomarkers of fish exposure to organic contaminants. In this work, swordfish liver sections obtained from the Mediterranean Sea, the South African coasts (South Atlantic and South Western Indian Oceans) and the Central North Pacific Ocean were immunostained with antisera against CYP1A, Zrp, and Vtg. CYP1A induction was found in hepatocytes, epithelium of the biliary ductus and the endothelium of large blood vessels of fish from the Mediterranean Sea and South African waters, but not from the Pacific Ocean. Zrp and Vtg were immunolocalized in hepatocytes of male swordfish from the Mediterranean Sea and from South African waters. Plasma Dot-Blot analysis, performed in Mediterranean and Pacific specimens, revealed the presence of Zrp and Vtg in males from Mediterranean but not from Pacific. These results confirm previous findings about the potential exposure of Mediterranean swordfish to endocrine, disrupting chemicals and raise questions concerning the possible presence of xenobiotic contaminants off the Southern coasts of South Africa in both the South Atlantic and South Western Indian Oceans.

Characterizing the inhibitory action of zinc oxide nanoparticles on allergic-type mast cell activation.

The development of nanoparticles (NPs) for commercial products is undergoing a dramatic expansion. Many sunscreens and cosmetics now use zinc oxide (ZnO) or titania (TiO2) NPs, which are effective ultraviolet (UV) filters. Zinc oxide topical creams are also used in mild anti-inflammatory treatments. In this study we evaluated the effect of size and dispersion state of ZnO and TiO2 NPs, compared to "bulk" ZnO, on mast cell degranulation and viability. ZnO and TiO2 NPs were characterized using dynamic light scattering and disc centrifugation. Rat basophilic leukaemia (RBL-2H3) cells and primary mouse bone marrow-derived mast cells (BMMCs) were exposed to ZnO and TiO2 NPs of different sizes (25-200 nm) and surface coatings at concentrations from 1 to 200 µg/mL. The effect of NPs on immunoglobulin E (IgE)-dependent mast cell degranulation was assessed by measuring release of both ß-hexosaminidase and histamine via colorimetric and ELISA assays. The intracellular level of Zn(2+) and Ca(2+) ions were measured using zinquin ethyl ester and Fluo-4 AM fluorescence probes, respectively. Cellular viability was determined using the soluble tetrazolium-based MTS colorimetric assay. Exposure of RBL-2H3 and primary mouse BMMC to ZnO NPs markedly inhibited both histamine and ß-hexosaminidase release. This effect was both particle size and dispersion dependent. In contrast, TiO2 NPs did not inhibit the allergic response. These effects were independent of cytotoxicity, which was observed only at high concentrations of ZnO NPs, and was not observed for TiO2 NPs. The inhibitory effects of ZnO NPs on mast cells were inversely proportional to particle size and dispersion status, and thus these NPs may have greater potential than "bulk" zinc in the inhibition of allergic responses.

The effects of exogenous gonadotropins on ovarian adenylate cyclase activity.

Adenylate cyclase was assayed in pellets prepared by centrifuging for 10 min at 600 x g homogenates of ovaries of immature rats treated with PMS and hCG. Activity was detected in the absence of any stimulatory agents and increased markedly in the presence of fluoride. Dose-dependent activity occurred in vitro in response to LH ranging from 0.2 to 10 mug/ml of incubate but not at higher concentrations. Marked changes in adenylate cyclase activities were observed with preparations from ovaries excised at various times after gonadotropin treatment. These changes, measured as the response to both fluoride and LH were observed to occur in four main stages. An initial decrease occurred 1/2 to 1 day after administration of hCG. Activity then increased and remained significantly elevated from day 3 to 7. A second but more dramatic rise was observed on day 8 and this enhanced level of activity remained elevated until day 13. A decreased level of activity occurred on day 15. Unstimulated activity remained low for the 16 day period studied although significant rises were observed on day 3 and days 8 to 15 after the administration of hCG. We have suggested that these changes in the adenylate cyclase activity modulate the level of cyclic AMP in the luteal cells and thereby induce changes in the activity of enzymes involved in progestin biosynthesis.

Chorionic gonadotropin secretion by human embryos in vitro.

Embryos (n = 38) from an in vitro fertilization (IVF) program, unsuitable for transfer or cryopreservation, were maintained in culture for 14 days. The time course of hCG secretion into culture fluid was compared to observed embryo development. The earliest significant hCG rise was detected at day 7 post insemination for a partially hatched blastocyst, with hCG levels increasing 50-fold following blastocyst attachment to the surface of the culture vessel. Secretion of hCG was amplified by the addition of serum. There was no significant free alpha hCG or beta hCG secretion between days 6-12. We conclude that hCG production depends on the pathway of cytotrophoblast differentiation and commences with the appearance of the G1 phase in embryonic cells at the late blastocyst stage.

Decomposition of pharmacological activity indices into mutually independent components using principal component analysis.

Various subsets of pharmacological activity indices of benzodiazepines, of 8-quinolinol derivatives, and of rifamycin B amides were decomposed into mutually independent components by using principal component analysis. The activites not included into the subset were considered as the dependent variables. In three out of the six cases with a fair correlation coefficient (r less than or equal to 0.9) between pairs of primary pharmacological indices, the main component obtained by the decomposition procedure showed significantly higher correlation with the dependent variable than any of the original pharmacological activity indices. Factors, explaining a rather low portion of the total sample variance of the subset, may still account for important secondary effects.

Differential distribution of mRNA for the alpha- and beta-subunits of chorionic gonadotrophin in the implantation stage blastocyst of the marmoset monkey.

We studied the expression of mRNA encoding the alpha- and beta-subunits of marmoset chorionic gonadotrophin (mCG) in implantation stage blastocysts and in a trophoblastic cell line derived from such blastocysts. In this investigation in situ hybridization was carried out using digoxygenin-labelled riboprobes to localize the subunit transcripts. The trophoblastic cell line, known to secrete bioactive mCG, was used as a positive control. Marmoset uterine embryos were cultured to hatched blastocysts and following growth on Matrigel or plastic were processed for in situ hybridization at developmental stages ranging from 13-15 days post-conception. In serial sections mCG-beta mRNA was detected mainly in polar trophoblast. The mRNA for mCG-alpha was expressed more uniformly in polar and mural trophoblast. Transcripts for the beta-subunit were not expressed, or present as weak signals, in the inner cell mass (ICM) and endoderm. However, low levels of mRNA for mCG-alpha were detected in the ICM and visceral endoderm. We have concluded that mRNA for mCG-beta was primarily localized to patches of syncytiotrophoblast at the embryonic pole and sparsely distributed in mural trophoblast, while the transcripts for mCG-alpha were distributed more uniformly in differentiating cytotrophoblast and syncytium, and at much lower levels in ICM and early endoderm.

The fertilizability of human oocytes at different stages of meiotic maturation.

Human oocytes were collected from small follicles during natural menstrual cycles. The oocytes were cultured from six to 36 hours and were inseminated for four hours before termination of the maturation culture. Maturation and fertilizability was assessed from serial sections of individual oocytes. Germinal vesicle breakdown commenced after six hours. Metaphase I was attained by 40% of oocytes by 12 hours and by 60% at 18 hours. Metaphase II was observed in over 40% of oocytes after 24 hours of culture. Both the zona pellucida and vitellus were penetrable to sperm at all stages of meiosis. However, the penetrability of the vitellus increased significantly with advancing stages of meiotic maturity. The chromatin of sperm within germinal vesicle oocytes was not undergoing decondensation. Sperm chromatin was partially decondensed in metaphase I and completely swollen in metaphase II oocytes. Immature oocytes penetrated by sperm had retained their cortical granules whereas these granules were absent from fertilized, mature oocytes.

Aspects of multiple embryo transfer.

Three hundred seventy-two patients underwent laparoscopy for in vitro fertilization and embryo transfer. Of these, 156 were treated with clomiphene citrate alone, 203 with clomiphene citrate and hMG, and 13 with hMG alone. Two-hundred seventy-two of these patients underwent embryo transfer, and 55 pregnancies resulted. Of these, 30 were ongoing, 14 biochemical, 6 ectopic, and 5 aborted. Where four embryos were transferred, the pregnancy rate was found to be significantly higher than when a lesser number were transferred. When the embryos transferred were analyzed, however, it was found that fewer than 10% of all transferred embryos implanted and it was largely a function of multiple embryo transfer per patient that led to success. When the type of hyperstimulation was compared, clomiphene citrate cycles were found to be less successful in that they produced fewer oocytes and embryos than did cycles in which hyperstimulation was achieved by supplementary hMG. It is conceded that this is an overall analysis and that individuals, especially those producing twins, had more than one good embryo transferred into a receptive uterus. The data suggest that unsuccessful cycles had somewhat poorer quality embryos, nonreceptive endometria, or a combination of these factors when compared with those of the successful embryo transfer cycles. The only predictive factor as to outcome relates to the multiplicity of embryos transferred. The fact that some IVF cycles produce multiple pregnancies indicates that a predictor of embryo health is urgently required. However, until such a reliable predictor is obtained, multiple embryo transfer remains a major advance in the success of IVF and ET.

A zona opening procedure for improving in vitro fertilization at low sperm concentrations: a mouse model.

Micromanipulation was used for creating a small opening in the zona pellucida without damaging the ooplasm of oocytes. This was applied in mouse in vitro fertilization as a model for inseminating oocytes with low sperm concentrations. It was found that the opening in the zona did not influence the fertilization rate at 10(5) sperm/ml, but when the sperm concentration was decreased to 10(4)/ml, the oocytes containing the break had a significantly higher fertilization rate and blastocyst development, compared with their intact controls. The zona cracking procedure did not increase parthenogenetic activation nor polyspermic fertilization, although it did promote early hatching of some blastocysts. The birthrate of normal offspring was not affected by exposing oocytes to the zona opening process.

The surplus human embryo: its potential for growth, blastulation, hatching, and human chorionic gonadotropin production in culture.

Early embryos that were unsuitable for transfer to patients or for cryopreservation were cultured either in a human tubal fluid (HTF) or a minimum essential medium (MEM). A significantly higher proportion of embryos developed to blastocysts in MEM (26.8%) than in HTF (14.5%). Approximately similar proportions of embryos formed blastocysts in MEM in the presence or absence of serum. The rate of embryo growth to blastocysts was similar in all media. Blastocyst hatching occurred in MEM + or - serum, but it failed to occur in HTF with serum. Released human chorionic gonadotropin (hCG) from hatched and intrazonal blastocysts was detected by day 8 after fertilization. The mean amount of hCG produced by day 14 was 19,500 mIU from hatched and 1,550 mIU from intrazonal blastocysts. Serum stimulated the output of hCG.

The secretion of hormones during the culture of human preimplantation embryos with corona cells.

Following in vitro fertilization of human preovulatory oocytes from spontaneously ovulating women, determinations were made of the secretion into the culture medium of progesterone, estradiol, human chorionic gonadotropin (hCG), and the prostaglandins (PG) E2 and F2 alpha, over a 3- to 4-day period of embryo development. It was found that the corona cells associated with the egg could account, between days 2 and 3, for a mean daily secretion of 50 ng of progesterone and approximately 100 pg of estradiol, PGE2, and PGF2 alpha, respectively. Mechanical removal of the corona cells after about 48 hours for the examination of the egg for cleavage reduced the mean amount of progesterone produced on day 3 to 2 ng, and a concomitant decrease in estradiol. PGE2, and PGF2 alpha was observed. Steroid secretion could be restored on day 3 to more than 50% of that secreted on day 2 by returning some detached corona cells to the culture medium containing the embryo. hCG was not detected (less than 2 mIU/ml) in the culture medium at any stage of embryo culture over the 3- to 4-day period.

Effect of sperm antibodies in females on human in vitro fertilization.

The effect of sperm antibodies derived from the female partner's serum on fertilization and embryo cleavage was evaluated by analyzing the Royal Women's Hospital in vitro fertilization (IVF) data. The results suggest that antispermatozoal isoantibodies detected by the immunobead test (IBT) can interfere with IVF. Thus, in a group of patients with IBT-IgG and IBT-IgA sperm antibody titers of greater than or equal to 10 in serum, a low fertilization rate (15%) was obtained when the wife's serum was used as serum supplement in the IVF culture medium. Where replacement (antibody-negative donor or cord) serum was used in the culture medium, a higher fertilization rate (69%) was obtained (P less than 0.01). These results underline the importance of using replacement serum in cases where the wife has significant sperm antibody levels in her serum. Six pregnancies were obtained in the antibody-positive group (n = 20), five of which occurred in patients with IBT-IgG and IBT-IgA-titers less than 10, for a pregnancy rate of 5/9 in this subgroup. Four of these patients delivered (4/9). Analysis of larger groups of antibody-positive patients is required for further evaluation of these results and ascertainment of the likelihood of occurrence of posttransfer effects of sperm antibodies on the embryo.

Effects of human seminal plasma on fertilizing capacity of human spermatozoa.

Using zona-free hamster eggs and salt-stored human eggs for assessing the fertilizing capacity of human spermatozoa, the effects of human seminal plasma on fertilizing capacity of human spermatozoa were investigated. The persistent presence of seminal plasma prevented sperm attachment to and penetration into the zona pellucida and vitellus. A series of experiments with zona-free hamster eggs revealed that, once the spermatozoa were preincubated in a seminal plasma-free environment known to induce the acrosome reaction, the seminal plasma no longer interfered with sperm-egg fusion. The native seminal plasma appears to interfere with both the acrosome reaction and vigorous motility of the spermatozoa, and this could be the reason fertilization fails when the plasma is consistently present around the spermatozoa. The fertilization-disturbing factor or factors in the seminal plasma appear to be macromolecular substances or substances associated with macromolecules.