Robustly estimating the demographic contribution of immigration: Simulation, sensitivity analysis and seals.

Identifying important demographic drivers of population dynamics is fundamental for understanding life-history evolution and implementing effective conservation measures. Integrated population models (IPMs) coupled with transient life table response experiments (tLTREs) allow ecologists to quantify the contributions of demographic parameters to observed population change. While IPMs can estimate parameters that are not estimable using any data source alone, for example, immigration, the estimated contribution of such parameters to population change is prone to bias. Currently, it is unclear when robust conclusions can be drawn from them. We sought to understand the drivers of a rebounding southern elephant seal population on Marion Island using the IPM-tLTRE framework, applied to count and mark-recapture data on 9500 female seals over nearly 40 years. Given the uncertainty around IPM-tLTRE estimates of immigration, we also aimed to investigate the utility of simulation and sensitivity analyses as general tools for evaluating the robustness of conclusions obtained in this framework. Using a Bayesian IPM and tLTRE analysis, we quantified the contributions of survival, immigration and population structure to population growth. We assessed the sensitivity of our estimates to choice of multivariate priors on immigration and other vital rates. To do so we make a novel application of Gaussian process priors, in comparison with commonly used shrinkage priors. Using simulation, we assessed our model's ability to estimate the demographic contribution of immigration under different levels of temporal variance in immigration. The tLTRE analysis suggested that adult survival and immigration were the most important drivers of recent population growth. While the contribution of immigration was sensitive to prior choices, the estimate was consistently large. Furthermore, our simulation study validated the importance of immigration by showing that our estimate of its demographic contribution is unlikely to result as a biased overestimate. Our results highlight the connectivity between distant populations of southern elephant seals, illustrating that female dispersal can be important in regulating the abundance of local populations even when natal site fidelity is high. More generally, we demonstrate how robust ecological conclusions may be obtained about immigration from the IPM-tLTRE framework, by combining sensitivity analysis and simulation.

Reproductive phenology is a repeatable, heritable trait linked to the timing of other life-history events in a migratory marine predator.

Population-level shifts in reproductive phenology in response to environmental change are common, but whether individual-level responses are modified by demographic and genetic factors remains less well understood. We used mixed models to quantify how reproductive timing varied across 1772 female southern elephant seals (Mirounga leonina) breeding at Marion Island in the Southern Ocean (1989-2019), and to identify the factors that correlate with phenological shifts within and between individuals. We found strong support for covariation in the timing of breeding arrival dates and the timing of the preceding moult. Breeding arrival dates were more repeatable at the individual level, as compared with the population level, even after accounting for individual traits (wean date as a pup, age and breeding experience) associated with phenological variability. Mother-daughter similarities in breeding phenology were also evident, indicating that additive genetic effects may contribute to between-individual variation in breeding phenology. Over 30 years, elephant seal phenology did not change towards earlier or later dates, and we found no correlation between annual fluctuations in phenology and indices of environmental variation. Our results show how maternal genetic (or non-genetic) effects, individual traits and linkages between cyclical life-history events can drive within- and between-individual variation in reproductive phenology.

Trade-offs between age-related breeding improvement and survival senescence in highly polygynous elephant seals: Dominant males always do better.

Life history trade-off theory predicts that current reproduction can negatively affect survival and future reproduction. Few studies have assessed breeding costs for males of polygynous species compared to females, despite substantial variation in breeding success among individual males (e.g. subordinate cf. dominant breeders). Specifically, differentiating between the cost of attending breeding seasons, and the additional cost of successfully securing and mating females is lacking. We investigated whether trade-offs are present in the highly polygynous male southern elephant seal (Mirounga leonina) using 34-years of individual-level data. We compare age-specific survival, recruitment and future breeding success probabilities of pre-breeders (males yet to recruit) and breeders (subordinate and dominant social ranks) using multievent models. Pre-breeders and breeders of overlapping ages had similar survival probabilities, suggesting that there was no attendance cost for early recruits. In addition, the probability of recruiting as a dominant breeder never exceeded recruitment probability as a subordinate breeder of the same age. Therefore, older pre-breeders that delayed attendance costs generally did not improve their breeding success (probability of being dominant) at recruitment more than younger recruits. Rather, recruitment age may be a function of individual quality, with lower quality individuals requiring more time to socially mature. When comparing subordinate and dominant breeders, we found clear evidence for survival senescence, with subordinate breeders having a higher baseline mortality. In contrast, age-specific future breeding success (probability of being dominant at t + 1) increased with age, with dominant breeders maintaining higher subsequent breeding success than subordinate breeders. The opposite trends in survival and future breeding success for both subordinate and dominant breeders may indicate a lifetime, population-level trade-off. However, we found no evidence to suggest that being a dominant breeder consecutively (and having a higher accumulated breeding cost) accelerated the rate of senescence when compared to individuals that were previously subordinate. Thus, males experienced actuarial senescence regardless of social rank, with dominant (and possibly high quality) breeders showing a reduced trade-off between survival and future breeding success. We make several novel contributions to understanding polygynous male life histories and southern elephant seal demography.

Identifying prey capture events of a free-ranging marine predator using bio-logger data and deep learning.

Marine predators are integral to the functioning of marine ecosystems, and their consumption requirements should be integrated into ecosystem-based management policies. However, estimating prey consumption in diving marine predators requires innovative methods as predator-prey interactions are rarely observable. We developed a novel method, validated by animal-borne video, that uses tri-axial acceleration and depth data to quantify prey capture rates in chinstrap penguins (Pygoscelis antarctica). These penguins are important consumers of Antarctic krill (Euphausia superba), a commercially harvested crustacean central to the Southern Ocean food web. We collected a large data set (n = 41 individuals) comprising overlapping video, accelerometer and depth data from foraging penguins. Prey captures were manually identified in videos, and those observations were used in supervised training of two deep learning neural networks (convolutional neural network (CNN) and V-Net). Although the CNN and V-Net architectures and input data pipelines differed, both trained models were able to predict prey captures from new acceleration and depth data (linear regression slope of predictions against video-observed prey captures = 1.13; R 2 ≈ 0.86). Our results illustrate that deep learning algorithms offer a means to process the large quantities of data generated by contemporary bio-logging sensors to robustly estimate prey capture events in diving marine predators.

The effect of prey abundance and fisheries on the survival, reproduction, and social structure of killer whales (Orcinus orca) at subantarctic Marion Island.

Most marine apex predators are keystone species that fundamentally influence their ecosystems through cascading top-down processes. Reductions in worldwide predator abundances, attributed to environmental- and anthropogenic-induced changes to prey availability and negative interactions with fisheries, can have far-reaching ecosystem impacts. We tested whether the survival of killer whales (Orcinus orca) observed at Marion Island in the Southern Indian Ocean correlated with social structure and prey variables (direct measures of prey abundance, Patagonian toothfish fishery effort, and environmental proxies) using multistate models of capture-recapture data spanning 12 years (2006-2018). We also tested the effect of these same variables on killer whale social structure and reproduction measured over the same period. Indices of social structure had the strongest correlation with survival, with higher sociality associated with increased survival probability. Survival was also positively correlated with Patagonian toothfish fishing effort during the previous year, suggesting that fishery-linked resource availability is an important determinant of survival. No correlation between survival and environmental proxies of prey abundance was found. At-island prey availability influenced the social structure of Marion Island killer whales, but none of the variables explained variability in reproduction. Future increases in legal fishing activity may benefit this population of killer whales through the artificial provisioning of resources they provide.

Positive early-late life-history trait correlations in elephant seals.

Correlations between early- and late-life performance are a major prediction of life-history theory. Negative early-late correlations can emerge because biological processes are optimized for early but not late life (e.g., rapid development may accelerate the onset of senescence; "developmental theory of aging") or because allocation to early-life performance comes at a cost in terms of late-life performance (as in the disposable soma theory). But variation in genetic and environmental challenges that each individual has to cope with during early life may also lead to positive early-late life-history trait correlations (the "fixed heterogeneity" or "individual quality" hypothesis). We analyzed individual life-history trajectories of 7,420 known-age female southern elephant seals (Mirounga leonina) monitored over 36 yr to determine how actuarial senescence (a proxy for late-life performance) correlate with age at first reproduction (a proxy for early-life performance). As some breeding events may not be detected in this field study, we used a custom "multievent" hierarchical model to estimate the age at first reproduction and correlate it to other life-history traits. The probability of first reproduction was 0.34 at age 3, with most females breeding for the first time at age 4, and comparatively few at older ages. Females with an early age of first reproduction outperformed delayed breeders in all aspects we considered (survival, rate of senescence, net reproductive output) but one: early breeders appeared to have an onset of actuarial senescence 1 yr earlier compared to late breeders. Genetics and environmental conditions during early life likely explain the positive correlation between early- and late-life performance. Our results provide the first evidence of actuarial senescence in female southern elephant seals.

Determinants of moult haulout phenology and duration in southern elephant seals.

Phenological shifts are among the most obvious biological responses to environmental change, yet documented responses for Southern Ocean marine mammals are extremely rare. Marine mammals can respond to environmental changes through phenological flexibility of their life-history events such as breeding and moulting. Southern elephant seals (Mirounga leonina) undergo an obligatory annual moult which involves the rapid shedding of epidermal skin and hair while seals fast ashore. We quantified the timing (phenology) and duration (the time from arrival ashore to departure) of the moult haulout of 4612 female elephant seals at Marion Island over 32 years. Using linear mixed-effects models, we investigated age, breeding state and environmental drivers of moult timing and haulout duration. We found no clear evidence for a temporal shift in moult phenology or its duration. Annual variation in moult arrival date and haulout duration was small relative to age and breeding effects, which explained more than 90% of the variance in moult arrival date and 25% in moult haulout duration. All environmental covariates we tested explained minimal variation in the data. Female elephant seals moulted progressively later as juveniles, but adults age 4 and older had similar moult start dates that depended on the breeding state of the female. In contrast, moult haulout duration was not constant with age among adults, but instead became shorter with increasing age. Moulting is energetically expensive and differences in the moult haulout duration are possibly due to individual variation in body mass and associated metabolizable energy reserves, although other drivers (e.g. hormones) may also be present. Individual-based data on moult arrival dates and haulout duration can be used as auxiliary data in demographic modelling and may be useful proxies of other important biological parameters such as body condition and breeding history.

Abundance of Indo-Pacific bottlenose dolphins (Tursiops aduncus) along the south coast of South Africa.

Coastally distributed dolphin species are vulnerable to a variety of anthropogenic pressures, yet a lack of abundance data often prevents data-driven conservation management strategies from being implemented. We investigated the abundance of Indo-Pacific bottlenose dolphins (Tursiops aduncus) along the south coast of South Africa, from the Goukamma Marine Protected Area (MPA) to the Tsitsikamma MPA, between 2014 and 2016. During this period, 662.3h of boat-based photo-identification survey effort was carried out during 189 surveys. The sighting histories of 817 identified individuals were used to estimate abundance using capture-recapture modelling. Using open population (POPAN) models, we estimated that 2,155 individuals (95% CI: 1,873-2,479) occurred in the study area, although many individuals appeared to be transients. We recorded smaller group sizes and an apparent decline in abundance in a subset of the study area (Plettenberg Bay) compared to estimates obtained in 2002-2003 at this location. We recorded declines of more than 70% in both abundance and group size for a subset of the study area (Plettenberg Bay), in relation to estimates obtained in 2002-2003 at this location. We discuss plausible hypotheses for causes of the declines, including anthropogenic pressure, ecosystem change, and methodological inconsistencies. Our study highlights the importance of assessing trends in abundance at other locations to inform data-driven conservation management strategies of T. aduncus in South Africa.

Making use of multiple surveys: Estimating breeding probability using a multievent-robust design capture-recapture model.

Increased environmental stochasticity due to climate change will intensify temporal variance in the life-history traits, and especially breeding probabilities, of long-lived iteroparous species. These changes may decrease individual fitness and population viability and is therefore important to monitor. In wild animal populations with imperfect individual detection, breeding probabilities are best estimated using capture-recapture methods. However, in many vertebrate species (e.g., amphibians, turtles, seabirds), nonbreeders are unobservable because they are not tied to a territory or breeding location. Although unobservable states can be used to model temporary emigration of nonbreeders, there are disadvantages to having unobservable states in capture-recapture models. The best solution to deal with unobservable life-history states is therefore to eliminate them altogether. Here, we achieve this objective by fitting novel multievent-robust design models which utilize information obtained from multiple surveys conducted throughout the year. We use this approach to estimate annual breeding probabilities of capital breeding female elephant seals (Mirounga leonina). Conceptually, our approach parallels a multistate version of the Barker/robust design in that it combines robust design capture data collected during discrete breeding seasons with observations made at other times of the year. A substantial advantage of our approach is that the nonbreeder state became "observable" when multiple data sources were analyzed together. This allowed us to test for the existence of state-dependent survival (with some support found for lower survival in breeders compared to nonbreeders), and to estimate annual breeding transitions to and from the nonbreeder state with greater precision (where current breeders tended to have higher future breeding probabilities than nonbreeders). We used program E-SURGE (2.1.2) to fit the multievent-robust design models, with uncertainty in breeding state assignment (breeder, nonbreeder) being incorporated via a hidden Markov process. This flexible modeling approach can easily be adapted to suit sampling designs from numerous species which may be encountered during and outside of discrete breeding seasons.

Dive behaviour and foraging effort of female Cape fur seals Arctocephalus pusillus pusillus.

While marine top predators can play a critical role in ecosystem structure and dynamics through their effects on prey populations, how the predators function in this role is often not well understood. In the Benguela region of southern Africa, the Cape fur seal (Arctocephalus pusillus pusillus) population constitutes the largest marine top predator biomass, but little is known of its foraging ecology other than its diet and some preliminary dive records. Dive information was obtained from 32 adult females instrumented with dive recorders at the Kleinsee colony (29°34.17' S, 16°59.80' E) in South Africa during 2006-2008. Most dives were in the depth range of epipelagic prey species (less than 50 m deep) and at night, reflecting the reliance of Cape fur seals on small, vertically migrating, schooling prey. However, most females also performed benthic dives, and benthic diving was prevalent in some individuals. Benthic diving was significantly associated with the frequency with which females exceeded their aerobic dive limit. The greater putative costs of benthic diving highlight the potential detrimental effects to Cape fur seals of well-documented changes in the availability of epipelagic prey species in the Benguela.

Differences in the association of PAI-1 activity with the metabolic syndrome between African and Caucasian women.

BACKGROUND AND AIMS: The association between PAI-1(act) and markers of the metabolic syndrome is well established in Caucasian populations, but data on African subjects is lacking. The aim of this study was to investigate possible differences between the association of PAI-1(act) and markers of the metabolic syndrome in Caucasian and African women. METHODS AND RESULTS: Cross-sectional data were collected from 95 African and 114 Caucasian women in the Potchefstroom district of the North West Province, South Africa. Plasma PAI-1(act) was almost twice as high in Caucasians compared to Africans (10.2 versus 5.2 U/mL, p<0.001). Correlations between markers of the metabolic syndrome and PAI-1(act) were remarkably stronger in Caucasians than in Africans. In multivariate regression analyses 56% of the variance of PAI-1(act) could be explained by metabolic syndrome variables in the Caucasian group compared to 12% in the African women. Waist circumference was the strongest independent predictor of PAI-1(act) in both groups. CONCLUSION: This study showed lower PAI-1(act) in African than in Caucasian women, along with less associations of PAI-1(act) with markers of the metabolic syndrome in the African than in the Caucasian women. The role of PAI-1(act) in the metabolic syndrome may be less prominent in Africans than in Caucasians.

Correction: Return Customers: Foraging Site Fidelity and the Effect of Environmental Variability in Wide-Ranging Antarctic Fur Seals.

[This corrects the article DOI: 10.1371/journal.pone.0120888.].

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus).

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10-12 month lactation period, female fur seals suckle pups on shore for 2-3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 microm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (alphaS2-casein, beta-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell-matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of ß-casein (CSN2) and ß-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk.

Substitution of high monounsaturated fatty acid avocado for mixed dietary fats during an energy-restricted diet: effects on weight loss, serum lipids, fibrinogen, and vascular function.

OBJECTIVE: First, we wanted to dispel the myth that avocados are fattening and therefore should be avoided in energy-restricted diets. Second, we examined the effects of avocados, a rich source of monounsaturated fatty acids, as part of an energy-restricted diet on weight loss, serum lipids, fibrinogen, and vascular function in overweight and obese subjects. METHODS: Sixty-one free-living volunteers (13 men and 48 women), with body mass index of 32 +/- 3.9 kg/m(2) (mean +/- standard deviation) participated in this randomized, controlled, parallel study. Subjects were paired and randomly assigned to one of two groups. The experimental group consumed 200 g/d of avocado (30.6 g of fat), which substituted for 30 g of other mixed dietary fats such as margarine or oil, and the control group excluded avocado from their energy-restricted diet for 6 wk. Seven-day isoenergetic menus were planned according to mean energy requirements of both sexes to provide total energy intakes consisting of 30% fat, 55% carbohydrates, and 15% protein. Anthropometric measurements, physical activity, blood pressure, and arterial compliance were measured with standard methods at the beginning and end of the intervention. Fasting blood samples were drawn at the beginning and end of the intervention. RESULTS: Fifty-five subjects completed the study. The compliance rate to avocado intake in the experimental group was 94.6%. The percentage of plasma oleic acid increased significantly with the consumption of avocado in the experimental group, whereas a decrease was seen in the percentage of myristic acid from baseline to the end of the intervention in both groups but was significant only in the experimental group. Anthropometric measurements (body mass, body mass index, and percentage of body fat) decreased significantly in both groups during the study (P < 0.001), and the change was similar in both groups. Serum lipid concentrations (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triacylglycerols), fibrinogen, blood pressure, and arterial compliance did not change significantly within or between groups. CONCLUSION: The consumption of 200 g/d of avocado within an energy-restricted diet does not compromise weight loss when substituted for 30 g of mixed dietary fat. Serum lipid concentrations, plasma fibrinogen, arterial compliance, and systolic and diastolic blood pressures were not affected by weight loss or avocado intake.

Return customers: foraging site fidelity and the effect of environmental variability in wide-ranging antarctic fur seals.

Strategies employed by wide-ranging foraging animals involve consideration of habitat quality and predictability and should maximise net energy gain. Fidelity to foraging sites is common in areas of high resource availability or where predictable changes in resource availability occur. However, if resource availability is heterogeneous or unpredictable, as it often is in marine environments, then habitat familiarity may also present ecological benefits to individuals. We examined the winter foraging distribution of female Antarctic fur seals, Arctocephalus gazelle, over four years to assess the degree of foraging site fidelity at two scales; within and between years. On average, between-year fidelity was strong, with most individuals utilising more than half of their annual foraging home range over multiple years. However, fidelity was a bimodal strategy among individuals, with five out of eight animals recording between-year overlap values of greater than 50%, while three animals recorded values of less than 5%. High long-term variance in sea surface temperature, a potential proxy for elevated long-term productivity and prey availability, typified areas of overlap. Within-year foraging site fidelity was weak, indicating that successive trips over the winter target different geographic areas. We suggest that over a season, changes in prey availability are predictable enough for individuals to shift foraging area in response, with limited associated energetic costs. Conversely, over multiple years, the availability of prey resources is less spatially and temporally predictable, increasing the potential costs of shifting foraging area and favouring long-term site fidelity. In a dynamic and patchy environment, multi-year foraging site fidelity may confer a long-term energetic advantage to the individual. Such behaviours that operate at the individual level have evolutionary and ecological implications and are potential drivers of niche specialization and modifiers of intra-specific competition.

Both fish oil and olive oil lowered plasma fibrinogen in women with high baseline fibrinogen levels.

This double-blind, cross-over study with olive oil as placebo, examined the effect of a daily dosage of 6 g fish oil on cardiovascular risk markers of 20 healthy young volunteers (10 men, 10 women). Serum lipids and lipoproteins, and plasma coagulation and fibrinolytic enzymes, including fibrinogen concentrations and plasminogen activator inhibitor-1 (PAI-1) activity were measured at baseline and after 6-week supplementation of either fish or olive oil. The results showed that fish oil had an independent lowering effect on triglycerides and coagulation factors Vc and VIIc. Both fish and olive oil significantly raised PAI-1 levels and lowered plasma factor Xc and fibrinogen levels in the women, who had higher initial levels than the men. Mean fibrinogen levels of the women were lowered from 3.23 +/- 0.98 to 2.64 +/- 0.55 g/l and from 3.19 +/- 0.72 to 2.66 +/- 0.49 g/l by fish and olive oil respectively. This study raises the question whether a particular fatty acid or group of fatty acids, or another constituent of the oil such as vitamin E may be responsible for the fibrinogen lowering effect.

Contribution of nutrition to the health transition in developing countries: a framework for research and intervention.

The major focus of public health programs in developing populations is alleviating undernutrition. In South Africa, however, as in many other developing countries, the African population is experiencing rapid urbanization characterized by a double burden of disease in which noncommunicable diseases (NCD) become more prevalent and infectious diseases remain undefeated. The possible mechanisms through which nutrition contributes to the additional vulnerability to NCD experienced by developing populations are explored and research priorities in this area are identified.

Defining the valid hemizona assay: accounting for binding variability within zonae pellucidae and within semen samples from fertile males.

OBJECTIVE: To achieve a better understanding of the variability in sperm and oocyte binding capacities will optimize use of the hemizona assay (HZA) as a predictor of sperm function. DESIGN: Limitations of the HZA were more clearly delineated by current studies: (1) variability of sperm binding capacity of men over a 90-day interval; (2) variability of sperm binding using different oocytes; and (3) lower limits of the number of sperm bound from the fertile control in two laboratories. PATIENTS: Semen was obtained from proven fertile men and one subfertile individual. MAIN OUTCOME MEASURE: The number of sperm tightly bound to the hemizona were measured and compared. RESULTS: In the initial study, 6 fertile control men exhibited a similar degree of variability in zona binding when studied over a 90-day interval. Average sperm binding for individuals ranged from 68 to 127. Second, 3 of the 15 simultaneous assays showed very low numbers of sperm bound, indicating that 20% of the zonae had poor binding. Third, from 18 men who had 0% fertilization in an in vitro fertilization system using mature oocytes, evaluation of their sperm by HZA was performed. The sperm bound poorly and the 95% confidence interval was 20 sperm bound. Thus, the fertile controls should bind greater than 20 sperm to distinguish them from the infertile group in the HZA system resulting in a valid assay. CONCLUSIONS: With these guidelines, applications of the HZA may be made with greater reassurance of a valid bioassay of sperm fertilizing potential.