Sylvatic and domestic Trichinella spp. in wild boars; infectivity, muscle larvae distribution, and antibody response

Thirty-six wild boars were inoculated with Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (USSR), T. pseudospiralis (USA), T. pseudospiralis (AUST), Trichinella murrelli, Trichinella T6, and Trichinella nelsoni. The wild boars were killed at 5 and 10 wk postinoculation (PI), and the number of muscle larvae per g (lpg) of tissue was determined for 18 muscles or muscle groups. Five weeks PI, all Trichinella genotypes had established as muscle larvae, but their infectivity varied widely: T. spiralis established in high numbers (mean = 296 lpg), T. britovi, T. nelsoni, and 1 of the T. pseudospiralis genotypes (AUST) in moderate numbers (mean = 53-74 lpg), whereas the remaining genotypes were poorly infective (mean 2-16 lpg). Because of considerable weight gain of the wild boars, an estimated total larval burden (live weight x lpg) was calculated for each animal. The total larval burden did not change significantly over time for T. spiralis, T. murrelli, T. britovi, T. nelsoni, and T. pseudospiralis (USA and USSR), whereas a significant reduction could be demonstrated for T. nativa, Trichinella T6, and T. pseudospiralis (AUST). Diaphragm and tongue were predilection sites in wild boars, independent of Trichinella genotype and infection level. At low infection levels, a greater percentage of larvae were found in diaphragm and tongue at 10 wk than 5 wk PI. Antibody responses increased rapidly between weeks 3 and 5 PI. For T. spiralis and T. nelsoni, the high antibody level persisted throughout the experimental period, but for T. nativa, T. britovi, T. murrelli, or Trichinella T6, the levels declined. For T. pseudospiralis, the antibody response increased more gradually between weeks 3 to 10 PI. Infection with all genotypes of Trichinella were detected using any of 7 excretory-secretory antigens, which points to the potential use of 1 common antigen for epidemiological studies on Trichinella in wild boars. In conclusion, T. spiralis is highly infective to wild boars, T. britovi, T. nelsoni, T. pseudospiralis (USA), and T. pseudospiralis (USSR) are moderately infective, and T. nativa, T. murrelli, T. pseudospiralis (AUST), and Trichinella T6 are poorly adapted to this host species.     

Taxonomic revision of the genus Trichinella.

The analysis of genetic, biochemical, and biological data on about 300 Trichinella isolates, reported in the literature, allows a taxonomic revision of this genus. We propose the recognition of 5 sibling species, Trichinella spiralis (Owen, 1835) sensu stricto; Trichinella nativa Britov and Boev, 1972; Trichinella pseudospiralis Garkavi, 1972; Trichinella nelsoni Britov and Boev, 1972 sensu stricto; and Trichinella britovi n. sp., on the basis of biochemical and biological characteristics. Trichinella britovi n. sp. is characterized by distribution in the Palaearctic Region; newborn larvae (NBL) production in vitro of 35-55 NBL/72 hr; nurse cell development time (NC d.t.) between 24 and 42 days postinfection (d.p.i.); low reproductive capacity index (RCI) in mice, rats, and pigs; low resistance to freezing; 1 unique marker allozyme; and moderate pathogenicity for humans. The new species is most similar to Trichinella nativa but differs from it in 4 allozymes, in having less resistance to freezing, in having a different pattern of major ribosomal DNA fragments after endonuclease digestion, and in distribution area. Trichinella nativa is characterized by a holarctic distribution; hosts that are sylvatic mammals; NBL production in vitro 28-54/72 hr; NC d.t. between 20 and 30 d.p.i.; low RCI in mice, rats, and pigs; high resistance to freezing; 2 unique marker allozymes; and moderate to severe pathogenicity for humans. Trichinella spiralis sensu stricto is characterized by a cosmopolitan distribution in domestic pigs, associated wildlife, and humans; high NBL production in vitro (greater than 90 NBL/72 hr); NC d.t. between 16 and 37 d.p.i.; high RCI in mice, rats, and pigs; no resistance to freezing; 6 unique marker allozymes; and high pathogenicity for humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidative,heat and anthelminthic stress responses in four species of Trichinella: comparative study

The aim of this study was to compare levels of stress proteins in four Trichinella species when exposed to different stressors. Heat shock protein (HSP) 60, 70 and 90 responses were evaluated in infective larvae (L(1)) of four classic Trichinella species following exposure to oxidative, anthelminthic and thermal stress. Larvae of T. nativa, T nelsoni, T. pseudospiralis and T. spiralis were exposed to peroxide shock (0.2%, 1%, or 2% H(2)O(2)for 2h), high temperatures (40 degrees C or 45 degrees C for 2h), or 0.1 microg/ml of the benzimidazole anthelminthics: mebendazole (MBZ), albendazole (ALB) or thiabendazole (TBZ) for 4h. Following exposures, the L(1) were tested for induced morphological changes. Those observed were: (i) no change (in all species exposed to 40 degrees C) (ii) aberrant forms (in all species exposed to anthelminthics, in T. nativa, T. nelsoni and T. spiralis exposed to 45 degrees C, and in T. spiralis and T. nelsoni exposed to 0.2% H(2)O(2)) and (iii) severe degradation or death (in T. nativa and T. pseudospiralis exposed to 0.2% H(2)O(2), and in all species at 1% and 2% H(2)O(2)). In Western blot analyses, L(1) proteins were probed with monoclonal antibodies (mAbs) specific for the three HSPs. Greater changes in HSP levels occurred following H(2)O(2) exposure than with other stresses in all Trichinella species, while accumulation of a 50 kDa HSP was only observed in T. spiralis and T. pseudospiralis. Anthelminthic stress only caused decreased HSP levels in T. nativa. Thermal stress caused no significant changes in the HSP response of any species. It is suggested that other stress proteins (e.g., glucose-regulated proteins) may be involved in adaptation to thermal stress.     

Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs

Groups of pigs were inoculated with genotypes of Trichinella belonging to: Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (from Caucasus), T. pseudospiralis (from USA), Trichinella murrelli, Trichinella sp. (from North America), and Trichinella nelsoni. The pigs were sacrificed between 5 and 40weeks p.i., and the number of muscle larvae per gram (l.p.g.) of tissue was determined as an average of 18 muscles. All Trichinella genotypes were infective for pigs, but both their infectivity and persistence varied: 5weeks p.i., T. spiralis muscle larvae were present in high numbers (mean=427l.p.g.), while T. britovi, T. nelsoni, and T. pseudospiralis larvae were present in moderate numbers (means=24-52l.p.g.); larvae of the remaining genotypes were recovered only in low numbers (means=0.05-5. 00l.p.g.). The total larval burden (live weight of pigxl.p.g.) was constant over time for T. spiralis, T. britovi, and T. nelsoni, but declined significantly (P<0.05) for the other genotypes. Antibody responses could be detected 3-4weeks p.i. by seven different Trichinella ES antigens, but the antibody levels and dynamics differed significantly among the experimental groups. In pigs inoculated with T. spiralis, T. britovi, or T. nelsoni, the antibody level increased rapidly between weeks 3 and 5 p.i. and was stable or increased slightly throughout the experimental period. In pigs inoculated with T. nativa, T. murrelli, or Trichinella (T6) (from North America), a rapid increase was detected between weeks 3 and 5 p.i., but for these genotypes a reduction in the antibody levels was seen thereafter. In the pigs inoculated with T. pseudospiralis, the antibody level increased more gradually over a period from week 3 p. i. to weeks 15-20 p.i., and decreased thereafter. In general, all species of Trichinella were detected by any of the seven ES antigens, which points to the potential use of one common antigen for surveillance and epidemiological studies on both domestic and sylvatic Trichinella in pigs. Homologous ES antigens were slightly more sensitive in detecting antibodies to the corresponding Trichinella species.     

Tolerance to low temperatures of domestic and sylvatic Trichinella spp. in rat muscle tissue

The present study was designed to investigate the tolerance to low temperatures of 9 Trichinella isolates in rat muscle tissue. Nine groups of 24 rats were infected with encapsulated Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella murrelli, Trichinella T6, Trichinella nelsoni, and 3 nonencapsulated Trichinella pseudospiralis strains. Six rats from each of the groups were necropsied at 5, 10, 20, and 40 wk postinfection (wpi). Muscle tissues containing Trichinella larvae were exposed to temperatures of -18, -5, and 5 C for 1 or 4 wk, and afterward the reproductive capacity index (RCI) in mice was determined for the 9 individual Trichinella isolates. Only T. nativa muscle larvae were infective after freezing at a temperature of -18 C. At 5 wpi all encapsulated isolates, except for the tropical species T. nelsoni, remained infective after exposure to a temperature of -5 C for both 1 and 4 wk, whereas nonencapsulated T. pseudospiralis survived only 1 wk of exposure. All Trichinella spp. remained infective after exposure to a temperature of 5 C. Muscle larvae for all investigated species remained infective as long as they persisted in live rats during the experiment. Analysis of variance showed a significant effect of age on the temperature tolerance of encapsulated T. spiralis and nonencapsulated T. pseudospiralis. In addition, significant interaction between age of muscle larvae and length of exposure was found. In general Trichinella muscle larvae of medium age (10 and 20 wpi) tolerated freezing better than early and late stages of infection (5 and 40 wpi). This is the first study to demonstrate such a relationship between age of infection and temperature tolerance of Trichinella spp. muscle larvae.     

Biological variation in Trichinella species and genotypes

At present, the genus Trichinella comprises seven species of which five have encapsulated muscle larvae (T. spiralis, T. nativa, T. britovi, T. nelsoni and T. murrelli) and two do not (T. pseudospiralis and T. papuae) plus three genotypes of non-specific status (T6, T8 and T9). The diagnostic characteristics of these species are based on biological, biochemical and genetic criteria. Of biological significance is variation observed among species and isolates in parameters such as infectivity and immunogenicity. Infectivity of Trichinella species or isolates is determined, among other considerations, by the immune status of the host in response to species- or isolate-specific antigens. Common and particular antigens determine the extent of protective responses against homologous or heterologous challenge. The kinetics of isotype, cytokine and inflammatory responses against T. spiralis infections are isolate-dependent. Trichinella spiralis and T. pseudospiralis induce different dose-dependent T-cell polarizations in the early host response, with T. spiralis initially preferentially promoting Th1-type responses before switching to Th2 and T. pseudospiralis driving Th2-type responses from the outset.     

Characterization of a noncyst-forming isolate of Trichinella from a wild boar in Yugoslavia.

An isolate of Trichinella obtained from a wild boar in Yugoslavia did not form cysts in the musculature of its natural host. Subsequent inoculation into experimental hosts demonstrated that some larvae became encysted only after extended time periods, whereas others remained unencapsulated. Histological staining of larvae in the musculature demonstrated no deposition of collagen typically seen for Trichinella spiralis spiralis, Trichinella spiralis nativa, or Trichinella spiralis nelsoni. The Yugoslavian isolate, given the name of Zagreb isolate after the University where it was first studied, had low infectivity for pigs and mice. Isozyme analysis demonstrated greater homology with T. s. nelsoni than with other subspecies of Trichinella. Restriction fragment length polymorphisms and dot blot analyses further demonstrated the distinctive nature of this isolate. These results suggest that lack of cyst formation might be characteristic of isolates other than those designated Trichinella pseudospiralis and that this character might be important in the classification of Trichinella.     

Identification of Trichinella isolates by polymerase chain reaction--restriction fragment length polymorphism of the mitochondrial cytochrome c-oxidase subunit I gene.

We developed a polymerase chain reaction based approach using restriction fragment length polymorphisms of the mitochondrial cytochrome c-oxidase subunit I to identify nine genotypes (Trichinella spiralis, Trichinella britovi-European strains, Trichinella britovi-Japanese strains, Trichinella nativa, Trichinella nelsoni, Trichinella T5, Trichinella T6, Trichinella T8 and Trichinella pseudospiralis) in the genus Trichinella. Partial mitochondrial cytochrome c-oxidase subunit I genes of nine genotypes were amplified by polymerase chain reaction, sequenced, and digested with three restriction endonucleases (Mse I, Alu I and Bsp1248 I). This polymerase chain reaction based restriction fragment length polymorphism method allowed the identification of Trichinella genotypes. Trichinella spiralis, Trichinella britovi-Japanese strains, Trichinella nelsoni, T5 and Trichinella pseudospiralis were distinguishable by digestion with Mse I. Trichinella britovi-European strains and Trichinella T8 were distinguishable by digestion using Alu I, and Trichinella nativa and Trichinella T6 were distinguishable by double-digestion with Mse I and Bsp1286 I. The results obtained with this polymerase chain reaction based restriction fragment length polymorphism assay confirmed those previously reported by others and support the separation of the Japanese isolates as a new genotype, namely Trichinella T9.     

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.     

Comparative study on polypeptide patterns of larvae of Trichinella isolates by two-dimensional electrophoresis

The two-dimensional patterns (isoelectrofocusing-IEF/polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate-SDS) of S3 fractions of muscle larvae of four Trichinella isolates were compared. The comparative study concerned six groups of polypeptides. It was observed that the Garkavi isolate of Trichinella pseudospiralis was clearly different from the other isolates, and it showed the simplest IEF/SDS polypeptide pattern. The C-76 isolate of T. nelsoni had only four of the six groups, distinguishing it from the GM-1 isolate of T. spiralis and the Boev isolate of T. nativa that showed all the indicated groups.     

Freeze-resistant Trichinella (Trichinella nativa) Established on the Scandinavian Peninsula

Until the 1970’s, Trichinella spiralis (Owen 1835) was considered the only species within the genus Trichinella. Then T. pseudospiralis (Garkavi 1972) was classified as a separate species on the basis of morphological and biological features. The remaining morphologically homogenous “T. spiralis-group” has been split into 4 different species (or subspecies) on the basis of their biological and biochemical characteristics; T. nativa (Britov & Boev 1972), T. nelsoni (Britov & Boev 1972), T. spiralis sensu stricto and T. britovi (Pozio et al. 1992).     

Ribo HRM--detection of inter- and intra-species polymorphisms within ribosomal DNA by high resolution melting analysis supported by application of artificial allelic standards

Ribo HRM, a single-tube PCR and high resolution melting (HRM) assay for detection of polymorphisms in the large subunit ribosomal DNA expansion segment V, was developed on a Trichinella model. Four Trichinella species: T. spiralis (isolates ISS3 and ISS160), T. nativa (isolates ISS10 and ISS70), T. britovi (isolates ISS2 and ISS392) and T. pseudospiralis (isolates ISS13 and ISS1348) were genotyped. Cloned allelic variants of the expansion segment V were used as standards to prepare reference HRM curves characteristic for single sequences and mixtures of several cloned sequences imitating allelic composition detected in Trichinella isolates. Using the primer pair Tsr1 and Trich1bi, it was possible to amplify a fragment of the ESV and detect PCR products obtained from the genomic DNA of pools of larvae belonging to the four investigated species: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in a single tube Real-Time PCR reaction. Differences in the shape of the HRM curves of Trichinella isolates suggested the presence of differences between examined isolates of T. nativa, T. britovi and T. pseudospiralis species. No differences were observed between T. spiralis isolates. The presence of polymorphisms within the amplified ESV sequence fragment of T. nativa T. britovi and T. pseudospiralis was confirmed by sequencing of the cloned PCR products. Novel sequences were discovered and deposited in GenBank (GenBank IDs: JN971020-JN971027, JN120902.1, JN120903.1, JN120904.1, JN120906.1, JN120905.1). Screening the ESV region of Trichinella for polymorphism is possible using the genotyping assay Ribo HRM at the current state of its development. The Ribo HRM assay could be useful in phylogenetic studies of the Trichinella genus.     

Genetic diversity within the genus Trichinella as shown by cleavage fragment length polymorphism analysis

The genetic diversity within the genus Trichinella was studied using cleavage fragment length polymorphism (CFLP) analysis. The CFLP method generates specific fingerprints based on single nucleotide mutations. By this method the amplified intergenic regions of the 5S rRNA genes of the eight different genotypes of Trichinella were analysed. The CFLP pattern of T. spiralis was completely different compared with the sylvatic species T. britovi, T. nativa, T. nelsoni, and the genotypes Trichinella T5, Trichinella T6 and Trichinella T8. The T. pseudospiralis intergenic region can be differentiated by size from the other species of Trichinella.     

Trichinella murrelli n. sp: etiological agent of sylvatic trichinellosis in temperate areas of North America

Trichinella T5, collected from sylvatic carnivores in North America, was identified previously as a different phenotype of Trichinella, with an uncertain taxonomic level due to the availability of only 2 isolates. Cross-breeding experiments carried out with single female and male larvae of 2 strains of Trichinella T5, with single female and male larvae of 2 strains of Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis, Trichinella nelsoni, and Trichinella T6, showed a reproductive isolation of Trichinella T5. Viable offspring were obtained only when a female of Trichinella T5 was crossed with a male of T. britovi, but not vice versa. Furthermore, the analysis of biological, biochemical, and molecular data of 32 isolates collected from sylvatic animals in the Nearctic region and identified as Trichinella T5 permitted its reassessment at the species level. Trichinella murrelli n. sp. is characterized by the following: distribution in temperate areas of the Nearctic region; newborn larvae production in vitro of 29-36/72 hr; nurse cell development time between 24 and 70 days postinfection; reproductive capacity index in Swiss mice 1.2-9.5, in wild mice 29.5-159.8, in rats 0.7-2.4, and in pigs 0.03-0.0004; no resistance to freezing; ribosomal DNA fragments of 7.2 kb and/or 11.4 kb, plus 2.2 kb and 1.8 kb present after Dra I digested DNA when probed with total T. spiralis RNA; a specific amplicon of 179 bp after polymerase chain reaction (PCR) amplification with the primer set SB147G; a specific fragment of 1,600 bp after PCR amplification with the primer set Ts43CA and Hhb I digestion; long incubation period; and moderate to severe pathogenicity for humans. The new species is most similar to T. britovi, though it differs from T. britovi in the pattern of 2 allozymes, in the patterns of major ribosomal DNA and PCR-restriction fragment length polymorphism fragments, and in geographical distribution.     

Genetic differentiation of the Trichinella genus with isoenzymatic analysis

Twenty-six Trichinella isolates have been examined by the isoenzyme typing of ten enzyme systems (LDH, ME, 6PGDH, G6PDH, GOT, AK, PGM, ACON, MPI, GPI). Four different zymodemes were obtained. All the examined isolates have shown an electrophoretic behaviour like one or other of four reference strains. The isolates from Italy and Yugoslavia have an electrophoretic mobility like T. nelsoni reference strain. The isolates from France, Holland, Great Britain, Poland and USA have an electrophoretic mobility like T. spiralis reference strain. For T. nativa and T. pseudospiralis we have tested only the reference strains. These results support the validity of the taxonomy of Trichinella genus in four good species.     

Studies on the cryopreservation of Dictyocaulus viviparus (Nematoda) third-stage larvae

Various cooling (0.1-5,100 degrees C min-1) and warming (20-6,800 degrees C min-1) rates, stepped cooling schedules and four cryoprotective additives (dimethyl sulphoxide, methanol, ethanediol and glycerol) were investigated in cryopreservation studies with Dictyocaulus viviparus third-stage larvae. Exsheathment with sodium hypochlorite was essential to achieve significant survival. With uninterrupted cooling, highest survival (30% normally motile) was achieved with rates of 10-70 degrees C min-1. Survival was higher (50-75%) using 1 degree C min-1 to -10 degrees C followed by plunging into liquid nitrogen. The optimum warming rate was 6,800 degrees C min-1. The use of cryoprotectants led to marginally lower survival while varying the suspending media had no significant effect on survival. X-irradiated, exsheathed third-stage larvae cryopreserved by the optimum protocol yielded 38.3 +/- 4.2% survival. Two calves each infected with 45,000 (15,000 viable) exsheathed, unirradiated, cryopreserved third-stage larvae harboured 494 worms (1.1% infectivity) and 355 worms (0.8%) at necropsy. Numbers of first-stage larvae in the faeces reached 420/g and 105/g respectively 27 days after infection.     

Effects of rat and human intestinal lamina propria cells on viability and muscle establishment of Trichinella spiralis newborn larvae

Although eosinophils and other inflammatory cells from the circulation and peritoneal cavity can damage Trichinella spiralis newborn larvae (NBL) in vitro, the cytotoxic potential of cells from the intestinal lamina propria, a site that may be the first line of defense against NBL migration, is unknown. Accordingly, we examined the interaction between NBL and isolated intestinal lamina propria cells (ILPC), including an enriched eosinophil population, from rats and humans. Rat ILPC killed NBL in vitro only after a prolonged incubation of 6 days. However they strongly adhered to NBL after only 4 hr incubation and prevented muscle establishment of NBL injected intravenously. Human ILPC showed similar adherence as rat ILPC but no killing was seen at the incubation time tested (36 hr).     

Biological characterization of Trichinella isolates from various host species and geographical regions.

Forty isolates of Trichinella collected from 5 continents were compared for 7 biological characters: newborn larvae produced per female worm cultured in vitro at the seventh, eighth, and ninth day postinfection, host muscle nurse cell development time, reproductive capacity index in rats and chickens, and resistance of muscle larvae to freezing. The isolates also were compared by analyses of an environmental character of the location from which they were isolated: the isotherms for January and July. By factorial analysis of correspondence of the biological and environmental data, the 40 isolates were grouped into 8 gene pools (T1-T8). The environmental temperature-related distribution was more evident for the sylvatic isolates (T2, T3, T5, T6, T7, T8), than for T1, which was isolated from domestic pigs, and for T4, a bird-adapted, nonencapsulating genetic type. The 8 biological groups correlated closely with the 8 gene pools previously identified on the basis of allozyme analysis. These results support the concept that the genus Trichinella is composed of at least 5 distinct gene pools or sibling species: Trichinella spiralis sensu stricto (T1), Trichinella nativa (T2), Trichinella sp. (T3), Trichinella pseudospiralis (T4), and Trichinella nelsoni (T7), and 3 other groups of uncertain taxonomic status (i.e., T5, T6, and T8).     

Nonbeneficial effects of glycerol on the oocyte penetrating capacity of cryopreserved and incubated human spermatozoa

The effect of cryopreservation on human spermatozoa in the presence or absence of glycerol was assessed by using sperm motility, functional integrity of sperm membrane, and denuded hamster oocyte penetration tests. Glycerol treated cryopreserved spermatozoa yielded a significantly higher (P less than 0.01) percentage of motile sperm and percentage of sperm with functionally intact membrane immediately after thawing than the spermatozoa not treated with glycerol but cryopreserved. However, no significant difference was observed between these cryopreserved spermatozoa (either treated or untreated with glycerol) on the percentage of motile sperm and the rate of oocyte penetration when the sperm were washed and incubated for 2 hr in a medium containing no glycerol. Thus, it appears glycerol may not be beneficial, since cryopreservation of spermatozoa either treated or untreated with glycerol essentially yields similar oocyte-penetrating capacity of sperm.