Reinforcement of mate preference among hybridizing Heliconius butterflies.

Recent models of mate preference evolution suggest that direct selection on alleles at preference loci and correlated evolution of preference with locally adapted mating cues are more likely to drive the evolution of assortative mate preference than reinforcement. Mate preference evolution in mimetic Heliconius butterflies has been attributed to all three forms of selection, but here we show that reinforcement has been critical. By examining geographical variation in assortative mating and male mate preference among seven populations of three hybridizing Heliconius species from Costa Rica, we found pronounced character displacement of preference such that sexual isolation was enhanced in areas of interspecific contact. Of the different explanations for the evolution of assortative mate preference, only reinforcement is dependent on interspecific contact in this system. Thus, the observed pattern of reproductive character displacement of mate preference is best explained as a product of indirect selection generated by natural selection against nonmimetic hybrids.

Epididymal specificity and androgen regulation of rat EP2.

In primates, expression of the EP2 gene is androgen-dependent and epididymis-specific. EP2 mRNA expression was investigated in caput, corpus, and cauda regions of rat epididymis and in 15 other rat tissues. Polymerase chain reaction and Northern analyses showed that rat EP2 is expressed predominantly in the proximal caput epididymidis. EP2 mRNA expression was determined in proximal epididymides from castrated, sham-operated, and efferent duct-ligated rats. In castrated rats, EP2 mRNA decreased to <10% of that in sham-operated rats between Days 3 and 4 postcastration, demonstrating the androgen dependence of EP2 expression. In epididymides ligated unilaterally at the efferent ducts, EP2 mRNA levels were approximately equal to those in the unligated contralateral epididymides or in sham-operated rats, indicating that EP2 expression does not depend on testicular factors. In bilaterally castrated rats, immediate and delayed testosterone replacement showed the dependence of EP2 expression on circulating androgens. Injection of testosterone propionate (TP) on Days 0, 1, 2, and 3 postcastration maintained EP2 mRNA levels approximately equal to those in sham-operated rats. Starting at Day 4 postcastration, daily injection of TP for 7 days restored EP2 mRNA to approximately normal levels. These data indicate for the rat that EP2 is expressed specifically in the proximal caput epididymidis and that its expression depends on circulating androgens but not on testicular factors.

Effects of androgen deprivation on the histology of adult chimpanzee testis.

Until primate sperm are exposed to the unique microenvironment of the epididymis, they are not capable of fertilization or vigorous motility. Many of the proteins that contribute to the unique microenvironment of the primate epididymis, and thus to sperm maturation, are dependent on androgens to induce their synthesis and secretion. GnRH antagonists have proved effective in suppressing LH and testosterone synthesis and secretion, and thus in maintaining a state of androgen deprivation or functional hypogonadotropism. We report here the effects of GnRH antagonist-induced androgen-deprivation on the histology of the testicular interstitium and seminiferous epithelium of the adult male chimpanzee. After only 21 days of androgen-deprivation, chimpanzee testicular tissues exhibit specific atrophic changes, including the loss of contact between developing spermatocytes and between Sertoli cells and their developing spermatids, alterations in cell development resulting in missing maturation steps (elongating Sc and structurally complete Sd2 spermatids) and inappropriate cell associations, varying degrees of cytoplasmic degradation in germ cells, Sertoli cells, and Leydig cells, and a tubular lumen obscured by masses of sloughed primary and secondary spermatocytes and what appear histologically to be Sb1 and Sd1 spermatids.

Organization of the human gene encoding the epididymis-specific EP2 protein variants and its relationship to defensin genes.

The EP2 gene codes for at least nine message variants that are all specifically expressed in the epididymis. These variants putatively encode small secretory proteins that differ in their N- and C-termini, resulting in proteins that can have little or no sequence similarity to each other. We have isolated and sequenced the human EP2 gene to determine the molecular origin of these variants. The EP2 gene has two promoters, eight exons, and seven introns. Exons 3 and 6 encode protein sequences homologous to beta-defensins, a family of antimicrobial peptides. This sequence homology and the arrangement of promoters and defensin-encoding exons suggest that the EP2 gene originated from two ancestral beta-defensin genes arranged in tandem, each contributing a promoter and two exons encoding a leader sequence and a defensin peptide. The proposed evolutionary relationship between the EP2 gene and defensin genes is supported by the observation that the EP2 gene is located on chromosome 8p23 near the defensin gene cluster and is separated by 100 kilobases or less from DEFB2, the gene for beta-defensin-2. While the EP2 gene transcribes beta-defensin-like message variants, most of the known message variants code for nondefensin proteins or proteins containing only a partial defensin peptide sequence. We suggest that, during its evolution, the EP2 gene has acquired new functions that may be important for sperm maturation and/or storage in the epididymis.

Histological effects of androgen deprivation on the adult chimpanzee epididymis.

Primate sperm acquire functional maturity, including vigorous forward motility and the ability to fertilize an ovum, as they transit the unique, regional microenvironment of the epididymal lumen. Several proteins secreted into this luminal fluid are epididymal-specific and androgen-dependent, and thus contribute potentially to sperm maturation. For the adult male chimpanzee, we report the effects of GnRH antagonist-induced androgen deprivation on the histology of the epithelia and interstitium composing the ductuli efferentes, ductus epididymis, proximal ductus (vas) deferens. After 21 days of androgen deprivation, epididymal tissues exhibit characteristic atrophic changes, including cellular disorganization, degradation, and loss of structures. Androgen-deprived cytoplasm is differentially and characteristically disrupted, vacuolated, and reduced in volume, resulting in decreased epithelial height and loss of stereocilia. Most principal cell nuclei appear hyperchromatic, smaller in size, more irregular in outline, and disordered in arrangement, while others appear swollen and vacuolated. Apical cells of the efferent ducts and the basal cells and microvillar borders of the ductus epididymis seem minimally affected by androgen deprivation. Such histologically differential responses suggest correspondingly that androgen is differentially essential to the maintenance of the epididymis and thus to normal functioning of the component tissues. Therefore, epididymal epithelia directly and their secretions indirectly are differentially androgen-dependent.

Pitfall of an internal control plasmid: response of Renilla luciferase (pRL-TK) plasmid to dihydrotestosterone and dexamethasone.

The thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) is commonly used as a control for transfection efficiency in the Dual-Luciferase Reporter Assay System. While investigating hormone response elements in the promoters of the androgen-dependent, epididymis-specific EP2 gene, we found that hormone treatment affected the luciferase activity of pRL-TK-transfected cells. In African Green Monkey Kidney (CV-1) cells, cotransfected transiently with a hormone-responsive promoter-firefly luciferase reporter plasmid and with pRL-TK, Renilla luciferase activity increased in response to dihydrotestosterone (DHT) and decreased in response to dexamethasone (DEX). When a thromboxane synthase promoter Renilla luciferase plasmid (pRL-TS) was used in place of pRL-TK, Renilla luciferase activity remained constant in CV-1 cells treated with DHT but decreased in CV-1 cells treated with DEX. In transfection studies, internal control plasmid expression in response to treatment must be carefully monitored to ensure proper interpretation of normalized results.

Multiple promoter and splicing mRNA variants of the epididymis-specific gene EP2.

The EP2 gene codes for a family of androgen-dependent, epididymis-specific secretory proteins. Using probes derived from human HE2 cDNA, a chimpanzee epididymal cDNA library was screened. Five variants of chimpanzee EP2 cDNA were identified. Variant 1 (EP2A) is the chimpanzee ortholog of HE2. Variant 2 (EP2B) has an alternative 5' end. Variant 3 (EP2C) has an alternative 3' end. Two additional variants were identified by reverse transcriptase-polymerase chain reaction analysis. Variant 4 (EP2D) and variant 5 (EP2E) appear to lack an exon, resulting in a shift in the open reading frame. Presumably, the 5 variants originate from the same gene and result from alternative promoters and alternative splicing. Each of the putative proteins encoded by these variant messages has a leader sequence characteristic for a secretory protein. After removal of the leader sequence, each of these proteins is predicted to consist of 1 or 2 out of 4 possible peptide modules. Two of these modules have no recognizable homology to known proteins. The other 2 modules have a distribution of cysteine residues characteristic for beta-defensins, a family of proteins with antimicrobial activity.

Immunohistochemical localization of epididymal secretory glycoprotein EP1 in the adult male chimpanzee.

Proteins, synthesized by the epididymal epithelium, are secreted sequentially into the lumen of the ducts epididymis where they effect sperm maturation and enable functional motility and fertilizing capacity. EP1 is a major secretory glycoprotein of chimpanzee (Pan troglodytes) epididymis. The epididymal duct exhibits diverse histology (Smithwick & Young, 1997). Epithelia I-V of the efferent ducts show no characteristic anti-EP1 binding. The densest granules of anti-EP1 reaction product appear in epithelium VI adjacent to the basal lamina in the infranuclear region of the principal cells (PCs), in the cytoplasm of the apical half of the PCs, and in the perinuclear and perivacuolar cytoplasm of the basal cells. In epithelia VII-XIV of the ductus epididymis proper, anti-EP1 binding decreases distally and is localized in the cytoplasm of the PCs and basal cells, among the stereocilia of the luminal border, within various microvillar borders, and in the luminal fluid. Therefore, EP1 appears to be synthesized and secreted primarily in the caput region of the ductus epididymis and may be reabsorbed nonselectively across epithelia with apical microvilli, including the non-ciliated cells of efferent ducts, the distal corpus and cauda of the ductus epididymis, and the proximal ductus deferens.

HE2/EP2, an androgen-dependent protein from the epididymis of the chimpanzee, Pan troglodytes.

A cDNA clone of chimpanzee EP2 was obtained from epididymal RNA by RT-PCR and was sequenced. Chimpanzee EP2 and human HE2 cDNA were > 99% identical and the proteins were 100% identical. The derived amino acid sequence of chimpanzee EP2 showed a consensus sequence for a leader peptide typical of secreted proteins. Northern blot analysis indicated that expression of the gene encoding chimpanzee EP2 is specific to the epididymis and not to other chimpanzee tissues examined. RT-PCR and northern blot analysis of epididymides recovered from an androgen deprived adult male chimpanzee showed that the expression of EP2 is androgen dependent.

Sequential histology of the adult chimpanzee epididymis.

The chimpanzee epididymis consists of three gross anatomical regions: caput (Cp), corpus (Cr), and cauda (Cd). During epididymal transit, chimpanzee sperm undergo maturational changes (affecting motility, capacitation, and the acrosome reaction), which enable fertilization. Elucidation of the sequential histology of the chimpanzee epididymis is preliminary to determining the role of each region of epididymal duct in the process of sperm maturation. We report the histology, in the adult male chimpanzee, of sequential segments of epididymis from the proximal efferent ductules through the Cp, Cr, and Cd regions of the epididymis proper to the proximal ductus deferens. These data illustrate the gross architecture of the tissue and reveal the histology characteristic of the diverse epithelia composing the epididymal ducts at each level. The tubules composing the epididymis of the adult male chimpanzee present at least 16 histologically-distinct epithelia and their transitional forms. Such diversity of epithelia suggests a corresponding diversity of function. Although there are some clear differences, the histological appearance of the ducts, and of the component epithelia of the chimpanzee epididymis, is remarkably similar to that reported for the human.

Body composition and protein and fat accretion in various body components in growing gilts fed diets with different protein levels but estimated to contain similar levels of ideal protein.

A comparative slaughter technique was used to determine the effect of dietary protein levels on body composition and protein and fat accretion in various body components in Yorkshire gilts. Diets were formulated to contain similar levels of idea protein. Eight gilts were slaughtered at 20 kg BW to determine initial body composition. Twenty-four gilts were assigned to each of three dietary treatments (16.6, 15.0 and 13.0% CP) during the growing period (20 to 55 kg BW). During the finishing period (55 to 100 kg BW), dietary CP levels were reduced by approximately two percentage units. Eight gilts per treatment were slaughtered at 55 kg BW, and the remaining pigs were slaughtered at 100 kg BW. At slaughter, the pits were divided into three components: carcass, empty gastrointestinal tract, and other non-carcass parts. Half of the carcass was further dissected into retail cuts. None of the carcass retail cuts was affected (P > .10) by the dietary CP levels, except trimmed ham was smaller (P < .05) in gilts fed the 13% CP growing and 11% CP finishing diets. Far content in the empty body (P = .12) and in the carcass (P = .06) was highest and the empty body water accretion rate was lowest (P = .05) in gilts fed the 13% CP growing and 11% CP finishing diets. Other aspects of chemical body composition, protein and fat accretion rates, and estimated lean yields were not affected by dietary CP level (P > .10).

The effect of reducing excess dietary amino acids on growing-finishing pig performance: an elevation of the ideal protein concept.

Seventy-two gilts were used to determine the effect of reducing excess amino acid intake on growing-finishing pig performance. Separate diets were formulated for the growing (20 to 55 kg BW) and finishing (55 to 100 kg BW) periods. For each period, three diets were formulated that varied in total crude protein level and contained similar levels of digestible ideal protein. Crystalline amino acids were added to the low protein diets to overcome potential deficiencies. The grower diets contained .66% apparent ileal digestible lysine and contained 16.6, 15.0, and 13.0% CP, respectively; for the finisher diets, these values were .55% digestible lysine and 14.2, 12.8, and 11.0% CP, respectively. Amino acid analyses and a separate digestibility trial were conducted to confirm digestible amino acid levels in the dietary ingredients. During the growing and the overall growing-finishing period, daily gain, feed intake, and gain:feed were not affected (P > .10) by dietary treatment. Daily gain tended to be lower (P = .06) and gain:feed was poorer (P < .05) for pigs fed the 11.0% CP diet during the finishing phase. Although backfat thickness tended to be greater (P = .07) for pigs fed the lower-CP diet, estimated carcass lean yield was not affected (P > .10) by dietary treatment. Results of this study show that dietary CP can be reduced to 13% in the growing and 12.8% in the finishing diets of pigs as long as crystalline amino acids are added to match the apparent ileal digestible amino acid ratios in an assumed ideal protein.

Estimate of epididymal transit time in the chimpanzee.

During their passage through the epididymis, sperm undergo functional changes which result in their maturation and in their ability to fertilize ova. Maturational changes effected during epididymal transport are attributable to sequential changes in various regions of the plasmalemma of the sperm head and flagella. These functional changes in the plasmalemma result, at least in part, from the sequential binding of proteins secreted by the epididymal epithelium into the epididymal lumen. An estimate of epididymal transit time is essential to such investigations. Time elapsed from a testicular arterial infusion of a single pulse of tritiated-thymidine to the release of 3H-labeled sperm into the lumen of the seminiferous tubule was about 39 days. Seminal fluid-free 3H-labeled sperm first appear in the ejaculate about 41 +/- 1 days post-infusion. Total transit time for 3H-labeled Sd2 sperm released into the tubular lumen to appear in the ejaculate is estimated as the difference between these values. Since total transit time is equal to the seminiferous tubule transit time plus the epididymal transit time, epididymal transit time constitutes some lesser portion of the total transit time of 2 +/- 1 days.

Duration of spermatogenesis and relative frequency of each stage in the seminiferous epithelial cycle of the chimpanzee.

To determine the duration of 1 spermatogenic cycle, a single pulse of tritiated thymidine was infused into a branch of the spermatic artery in each of 3 chimpanzees (Pan troglodytes). Samples were recovered surgically prior to infusion, at 1 h, and at 3, 8, 14, 16, 17, 28, 30, 33, 40, 44, and 48 days postinfusion. Tissues were fixed in Bouin's solution, dehydrated, paraffin-embedded, sectioned at 5 micrometers, and stained. Pre-infusion samples were used in morphometric studies to estimate the percentage frequency of area occupied by each of the 6 cellular associations (stages I-VI) characteristic of chimpanzee spermatogenesis, and thus, to estimate the days duration of each stage. To estimate the duration of 1 spermatogenic cycle, pre- and post-infusion, tissue sections were coated with undiluted Kodak NTB2 liquid autoradiographic emulsion and incubated at 4 +/- 1 degree C. At optimum exposure times, slides were processed with Kodak D-19 and Fixer; light microscopic analyses were conducted to determine the most mature labeled cell in stage III for each of the sample times. The duration of the 6 stages (I-VI) are 4.2, 2.0, 4.3, 1.5, 1.3 and 0.6 days, respectively, and the duration of 1 spermatogenic cycle is approximately 14 days. Thus, the duration of spermatogenesis from the Ap spermatogonium to mature Sd2 spermatid is approximately 62.5 +/- 1.5 days or 4.46 spermatogenic cycles.

Molecular cloning and characterization of EPI-1, the major protein in chimpanzee (Pan troglodytes) cauda epididymal fluid.

A 27-kDa glycoprotein comprises approximately 20% of the total protein in chimpanzee (Pan troglodytes) cauda epididymal fluid. Polyclonal antibodies generated against this glycoprotein react with 27- and 25-kDa components in chimpanzee cauda epididymal fluid and in human, gorilla, chimpanzee, and monkey seminal fluid. According to microsequencing, the 27- and 25-kDa components (chimpanzee EPI-1) are identical to the cloned putative human epididymal protein HE1. Screening of a chimpanzee epididymal cDNA library enabled isolation of a cDNA clone of chimpanzee EPI-1. On the cDNA level, chimpanzee EPI-1 and human HE1 are 99% identical. Northern analysis localized chimpanzee EPI-1 mRNA to the distal caput epididymidis. With EPI-1 primers, polymerase chain reaction of reverse-transcribed rhesus monkey (Macaca mulatta) epididymal RNA enabled isolation of a rhesus monkey EPI-1 cDNA clone. The derived amino acid sequence of rhesus monkey EPI-1 is identical to chimpanzee EPI-1 and to human HE1. Northern analysis localized rhesus monkey EPI-1 mRNA to the distal caput and the proximal corpus epididymidis. Northern analysis also showed that chimpanzee EPI-1 and rhesus monkey EPI-1 gene products are expressed specifically in the epididymis and not in any other tissue examined.

Germ cell maturation and cellular associations in the seminiferous epithelial cycle of the chimpanzee.

Seminiferous tubule architecture, germ cell maturation steps and cellular associations (stages) of the spermatogenic cycle of the chimpanzee (Pan troglodytes) are resolved. Cross sections of seminiferous tubules usually exhibit 2 to 4 stages, occasionally 1, and rarely 5; stages are not functionally sequential in structurally contiguous regions. The cellular maturation steps are: dark type A stem cell (Ad), pale type A (Ap), type B(B) spermatogonia; resting or preleptotene (P1), leptotene (L), zygotene (Z), pachytene (P), diplotene (Di) primary spermatocytes; meiotic divisions (M1, M2); secondary spermatocytes (2 degrees S); six developmental stages of the spermatid (Sa, Sb1, Sb2, Sc, Sd1, Sd2) composing spermiogenesis. The germ cell maturation steps characteristic of the six cellular associations (stages I-VI) are: Ad, Ap, B, P, Sa, Sd1 (I); Ad, Ap, B, PI, P, Sa, Sd2 (II); Ad, Ap, B, PI, L, P, Sb1 (III); Ad, Ap, PI, L, P, Sb2 (IV); Ad, Ap, L, Z, P, Di, Sc (V); Ad, Ap, B, Z, P, Di, 2 degrees S, Sc (VI). Surgical pressure trauma causes sloughing of some 2 degrees S spermatocytes and some Sa, Sb1, Sb2, Sd1, and Sd2 spermatids, resulting in missing generations, and disrupts Sertoli cell attachments, affecting germ cell development and associations. In structure and function, chimpanzee spermatogenesis appears most similar to the human.

An economic evaluation of hospital-based hemodialysis and home-based peritoneal dialysis for pediatric patients.

The purpose of this study was to assess the relative health system cost of pediatric ambulatory hospital-based hemodialysis and home-based peritoneal dialysis, including both continuous ambulatory peritoneal dialysis and continuous cycling peritoneal dialysis when either treatment is equally appropriate. A cost analysis was performed from the viewpoint of the "study hospital" and service providers (physicians) using treatment protocols, based on current clinical practice, which incorporate procedures to establish dialysis access sites, ongoing dialysis maintenance, and possible complications. Cost estimates used information from the period between April 1, 1993, to March 31, 1994, including fully allocated inpatient and outpatient costs. A sensitivity analysis was conducted to analyze the effect of complications on treatment costs. Total annual costs (in 1994 Canadian dollars, $1.00 CDN approximately $0.75. US) of a typical and uncomplicated continuous ambulatory peritoneal dialysis, continuous cycling peritoneal dialysis, and hemodialysis patient were $47,569, $48,658, and $76,023, respectively. Differences in cost between peritoneal dialysis and hemodialysis patients were due to hemodialysis maintenance costs, which were attributed to larger physician fees (25.8 percent), greater direct treatment costs incurred by the study hospital (14.2 percent), and higher overhead costs (60.0 percent). The expected total cost of hemodialysis complicated by an arteriovenous fistula clot and central venous line blockages, or peritoneal dialysis complicated by hernia repair and peritonitis was $78,568 and $50,438 for hemodialysis and peritoneal dialysis, respectively. For the range of complication probabilities considered, expected total costs were always lower with peritoneal dialysis than with hemodialysis. The cost analysis demonstrates that peritoneal dialysis is less costly than hemodialysis for pediatric patients. Such analyses are but one component of the treatment decision, and as such, should not be viewed as the sole means to yield a treatment decision, but rather as a device for systematically evaluating the alternative treatment options.

Functional parameters of chimpanzee (Pan troglodytes) sperm from ejaculates collected by rectal probe electrostimulation and by artificial vagina.

This study compares functional parameters of sperm from ejaculates collected from 15 adult male chimpanzees using rectal probe electrostimulation (RPE) and from 10 adult male chimpanzees trained to use an artificial vagina (AV). Computer assisted motion analysis (CAMA) showed no significant differences in mean values for straight line velocity (VSL), linearity (LIN), curvilinear velocity (VCL), and lateral head movement (ALH) of sperm from ejaculates collected by RPE and by AV. There was, however, a significant difference (P < 0.01) in the population distribution for VSL and LIN, which indicates that sperm swim in a more convoluted manner in ejaculates collected by RPE than in ejaculates collected by AV. In the hamster zona-free ovum penetration assay (SPA), there were no significant differences in the percentages of hamster oocytes penetrated by sperm or in the number of sperm which penetrated each oocyte after 4 or 24 h incubation using sperm from ejaculates collected by RPE and by AV. Therefore, the lack of success using sperm from ejaculates collected by RPE to initiate pregnancy in the chimpanzee does not appear to result from abnormalities in sperm fertilizing capacity as measured in SPA. © 1996 Wiley-Liss, Inc.

Characteristics of chimpanzee (Pan troglodytes) ejaculates collected by rectal probe electrostimulation and by artificial vagina.

This study compares characteristics of ejaculates collected from 16 adult male chimpanzees using rectal probe electrostimulation (RPE) and from 10 adult male chimpanzees trained to use an artificial vagina (AV). Ejaculate weight, semen volume, and sperm number were significantly lower (P < 0.01) and percentage liquefaction was significantly higher (P < 0.01) in ejaculates collected by RPE. Percentages of motile sperm and of live sperm in semen did not differ significantly between the two collection methods. Total amounts of protein and of α-glucosidase activity were significantly lower (P < 0.01) in seminal fluid from RPE samples. For ejaculates collected by RPE, semen volume correlated positively with protein (r = 0.8640, P < 0.001), fructose (r = 0.6976, P < 0.001), and citrate (r = 0.6976, P < 0.001); sperm number correlated positively with α-glucosidase activity (r = 0.6547, P < 0.001); and protein correlated positively with fructose (r = 0.5906, P < 0.002), citrate (r = 0.5926, P < 0.002) and α-glucosidase activity (r = 0.6006, P < 0.001). For ejaculates collected by AV, semen volume correlated positively with percentage liquefaction (r = 0.6058, P < 0.001), protein (r = 0.8055, P < 0.001), fructose (r = 0.6606, P < 0.001), and citrate (r = 0.8272, P < 0.001); sperm number correlated positively with percentage of motile sperm (r = 0.4196, P 0.004); percentage of motile sperm correlated positively with percentage of live sperm (r = 0.4388, P < 0.002); and, protein correlated positively with fructose (r = 0.6947, P < 0.002) and with citrate (r = 0.5926, P < 0.002). These data show that there is a significant difference in semen parameters and in biochemical parameters of ejaculates obtained by RPE and by AV. © 1995 Wiley-Liss, Inc.

Effect of liquefaction time on semen parameters in chimpanzee (Pan troglodytes) ejaculates.

Ejaculates were obtained from eight adult male chimpanzees trained to use an artificial vagina. Spontaneously liquefied fractions were collected at 0-1, 1-2 and 2-4 h after ejaculation. The mean volumes of the spontaneously liquefied fractions did not differ significantly among the three fractions. The total number of sperm, the percentage of motile sperm and the percentage of live sperm decreased significantly (P < 0.005) between the 0-1 and 1-2 h fractions. Citrate concentration and alpha-glucosidase activity (mU/ml) decreased significantly (P < 0.005) from 0-1 to 1-2 to 2-4 h fractions. The volume of the liquefied fraction correlated positively (P < 0.001) with total protein and with total citrate at 0-1, 1-2 and 2-4 h. The total number of sperm correlated positively total with total alpha-glucosidase activity at 0-1, 1-2 and 2-4 h. Among the biochemical parameters measured, total protein and total citrate correlated positively at 0-1, 1-2 and 2-4 h. © 1995 Wiley-Liss, Inc.