The Dot-Probe Attention Bias Task as a Method to Assess Psychological Well-Being after Anesthesia: A Study with Adult Female Long-Tailed Macaques (Macaca fascicularis).

Understanding the impact routine research and laboratory procedures have on animals is crucial to improving their well-being and to the success and reproducibility of the research they are involved in. Cognitive measures of welfare offer insight into animals' internal psychological state, but require validation. Attention bias - the tendency to attend to one type of information over another - is a cognitive phenomenon documented in humans and animals that is known to be modulated by affective state (i.e., emotions). Hence, changes in attention bias may offer researchers a deeper perspective of their animals' psychological well-being. The dot-probe task is an established method for quantifying attention bias in humans (by measuring reaction time to a dot-probe replacing pairs of stimuli), but has yet to be validated in animals. We developed a dot-probe task for long-tailed macaques (Macaca fascicularis) to determine if the task can detect changes in attention bias following anesthesia, a context known to modulate attention and trigger physiological arousal in macaques. Our task included the following features: stimulus pairs of threatening and neutral facial expressions of conspecifics and their scrambled counterparts, two stimuli durations (100 and 1,000 ms), and counterbalancing of the dot-probe's position on the touchscreen (left and right) and location relative to the threatening stimulus. We tested 8 group-housed adult females on different days relative to being anesthetized (baseline and 1-, 3-, 7-, and 14-days after). At baseline, monkeys were vigilant to threatening content when stimulus pairs were presented for 100 ms, but not 1,000 ms. On the day immediately following anesthesia, we found evidence that attention bias changed to an avoidance of threatening content. Attention bias returned to threat vigilance by the third day postanesthesia and remained so up to the last day of testing (14-days after anesthesia). We also found that attention bias was independent of the type of stimuli pair (i.e., whole face vs. scrambled counterparts), suggesting that the scrambled stimuli retained aspects of the original stimuli. Nevertheless, whole faces were more salient to the monkeys as responses to these trials were generally slower than to scrambled stimulus pairs. Overall, our study suggests it is feasible to detect changes in attention bias following anesthesia using the dot-probe task in nonhuman primates. Our results also reveal important aspects of stimulus preparation and experimental design.

Comprehensive search filters for retrieving publications on nonhuman primates for literature reviews (filterNHP).

Nonhuman primates (NHPs) are widely studied across many scientific disciplines using a variety of techniques in diverse environments. Due to the wide scope of NHP research, substantial overlap in research topics and questions can occur, whose resulting scientific evidence is synthesized by literature reviews. Identifying all relevant research on a particular topic involving NHPs can be difficult and time consuming. By adopting objective search development techniques from systematic reviews, we developed search filters to detect all scientific publications involving NHPs in PubMed, PsycINFO (via EBSCOhost), and Web of Science. We compared the performance of our comprehensive NHP search filters to search strings typical of a novice database user (i.e., NHP simple search strings) and validated their sensitivity by combining these searches with a topic search of cortisol related studies. For all comparisons, our comprehensive NHP search filters retrieved considerably more scientific publications than the NHP simple search strings. Importantly, our comprehensive NHP search filters are easy to use (text can be copied and pasted into the database search engine) and detect the most recent publications that have yet to be indexed by the bibliographic databases queried. Additionally, we developed filterNHP, an R package and web-based application (https://filterNHP.dpz.eu), for researchers interested in literature searches involving a taxonomic sub-group of NHPs. filterNHP alleviates time necessary for adapting our comprehensive NHP search filters for a particular NHP sub-group by automatizing the creation of these search filters. Altogether, our comprehensive NHP search filters and those for taxonomic sub-groups generated by filterNHP will enable swift and easy retrieval of the available scientific literature involving NHPs, and thereby help enhance the quality of new NHP literature reviews that guide future scientific research (new experiments) and public policy (e.g., on welfare and conservation).

Parent-offspring facial resemblance increases with age in rhesus macaques.

Kin recognition is a key ability which facilitates the acquisition of inclusive fitness benefits and enables optimal outbreeding. In primates, phenotype matching is considered particularly important for the recognition of patrilineal relatives, as information on paternity is unlikely to be available via social familiarity. Phenotypic cues to both paternal and maternal relatedness exist in the facial features of humans and other primates. However, theoretical models suggest that in systems with uncertainty parentage it may be adaptive for offspring to conceal such cues when young, in order to avoid potential costs of being discriminated against by unrelated adults. Using experienced human raters, we demonstrate in a computer-based task that detection of parent-offspring resemblances in the faces of rhesus macaques (Macaca mulatta) increases significantly with offspring age. Moreover, this effect is specific to information about kinship, as raters were extremely successful at discriminating individuals even among the youngest animals. To our knowledge, this is the first evidence in non-humans for the age-dependent expression of visual cues used in kin recognition.

Female rhesus macaques discriminate unfamiliar paternal sisters in playback experiments: support for acoustic phenotype matching.

Widespread evidence exists that when relatives live together, kinship plays a central role in shaping the evolution of social behaviour. Previous studies showed that female rhesus macaques (Macaca mulatta) recognize familiar maternal kin using vocal cues. Recognizing paternal kin might, however, be more difficult as rhesus females mate promiscuously during the possible conception period, most probably concealing paternity. Behavioural observations indicate that semi free-ranging female rhesus macaques prefer to associate with their paternal half-sisters in comparison to unrelated females within the same group, particularly when born within the same age cohort. However, the cues and mechanism/s used in paternal kin discrimination remain under debate. Here, we investigated whether female rhesus macaques use the acoustic modality to discriminate between paternal half-sisters and non-kin, and tested familiarity and phenotype matching as the underlying mechanisms. We found that test females responded more often to calls of paternal half-sisters compared with calls of unrelated females, and that this discrimination ability was independent of the level of familiarity between callers and test females, which provides, to our knowledge, the first evidence for acoustic phenotype matching. Our study strengthens the evidence that female rhesus macaques can recognize their paternal kin, and that vocalizations are used as a cue.

Choice-based severity scale (CSS): assessing the relative severity of procedures from a laboratory animal's perspective.

One primary goal of laboratory animal welfare science is to provide a comprehensive severity assessment of the experimental and husbandry procedures or conditions these animals experience. The severity, or degree of suffering, of these conditions experienced by animals are typically scored based on anthropocentric assumptions. We propose to (a) assess an animal's subjective experience of condition severity, and (b) not only rank but scale different conditions in relation to one another using choice-based preference testing. The Choice-based Severity Scale (CSS) utilizes animals' relative preferences for different conditions, which are compared by how much reward is needed to outweigh the perceived severity of a given condition. Thus, this animal-centric approach provides a common scale for condition severity based on the animal's perspective. To assess and test the CSS concept, we offered three opportunistically selected male rhesus macaques (Macaca mulatta) choices between two conditions: performing a cognitive task in a typical neuroscience laboratory setup (laboratory condition) versus the monkey's home environment (cage condition). Our data show a shift in one individual's preference for the cage condition to the laboratory condition when we changed the type of reward provided in the task. Two additional monkeys strongly preferred the cage condition over the laboratory condition, irrespective of reward amount and type. We tested the CSS concept further by showing that monkeys' choices between tasks varying in trial duration can be influenced by the amount of reward provided. Altogether, the CSS concept is built upon laboratory animals' subjective experiences and has the potential to de-anthropomorphize severity assessments, refine experimental protocols, and provide a common framework to assess animal welfare across different domains.

A Touchscreen-Based, Multiple-Choice Approach to Cognitive Enrichment of Captive Rhesus Macaques (Macaca mulatta).

Research on the psychological and physiological well-being of captive animals has focused on investigating different types of social and structural enrichment. Consequently, cognitive enrichment has been understudied, despite the promising external validity, comparability, and applicability. As we aim to fill this gap, we developed an interactive, multiple-choice interface for cage-mounted touchscreen devices that rhesus monkeys (Macaca mulatta) can freely interact with, from within their home enclosure at the Cognitive Neuroscience Laboratory of the German Primate Center. The multiple-choice interface offers interchangeable activities that animals can choose and switch between. We found that all 16 captive rhesus macaques tested consistently engaged with the multiple-choice interface across 6 weekly sessions, with 11 of them exhibiting clear task preferences, and displaying proficiency in performing the selected tasks. Our approach does not require social separation or dietary restriction and is intended to increase animals' sense of competence and agency by providing them with more control over their environment. Thanks to the high level of automation, our multiple-choice interface can be easily incorporated as a standard cognitive enrichment practice across different facilities and institutes working with captive animals, particularly non-human primates. We believe that the multiple-choice interface is a sustainable, scalable, and pragmatic protocol for enhancing cognitive well-being and animal welfare in captivity.

Group size and mating system predict sex differences in vocal fundamental frequency in anthropoid primates.

Vocalizations differ substantially between the sexes in many primates, and low-frequency male vocalizations may be favored by sexual selection because they intimidate rivals and/or attract mates. Sexual dimorphism in fundamental frequency may be more pronounced in species with more intense male mating competition and in those with large group size, where social knowledge is limited and efficient judgment of potential mates and competitors is crucial. These non-mutually exclusive explanations have not been tested simultaneously across primate species. In a sample of vocalizations (n = 1914 recordings) across 37 anthropoid species, we investigated whether fundamental frequency dimorphism evolved in association with increased intensity of mating competition (H1), large group size (H2), multilevel social organization (H3), a trade-off against the intensity of sperm competition (H4), and/or poor acoustic habitats (H5), controlling for phylogeny and body size dimorphism. We show that fundamental frequency dimorphism increased in evolutionary transitions towards larger group size and polygyny. Findings suggest that low-frequency male vocalizations in primates may have been driven by selection to win mating opportunities by avoiding costly fights and may be more important in larger groups, where limited social knowledge affords advantages to rapid assessment of status and threat potential via conspicuous secondary sexual characteristics.

Toward a Standardized Test of Fearful Temperament in Primates: A Sensitive Alternative to the Human Intruder Task for Laboratory-Housed Rhesus Macaques (Macaca mulatta).

Standardized and sensitive tests to assess differences in temperament among primates housed in captivity are essential for monitoring welfare and improving science outcomes through reduced noise in data. Fearful temperament in primates has traditionally been assessed using the Human Intruder Test (HIT) in which duration of bodily freeze in response to approach by an unknown human is measured. The HIT is susceptible to variation between facilities in execution, interpretation of data and could be stressful for animals with more fearful temperaments. We tested the applicability of a touch-screen task with putatively negative stimuli as a more standardizable and sensitive tool for measuring fearful temperament in laboratory primates. Seventeen adult male rhesus macaques were assessed for fearfulness using the HIT. They were then tested on a touch-screen task designed to measure two behavioral indices of fearfulness: behavioral inhibition and response-slowing. We predicted monkeys assessed as having more fearful temperament in the HIT, would show the greatest degree of behavioral inhibition and response-slowing to negative pictures in the touch-screen task. In Study 1, monkeys were rewarded with juice for touching gray squares on the screen (control trials). On test trials a picture of an unknown male conspecific face with direct-gaze (signaling threat) was shown. Monkeys were less likely to touch direct-gaze faces than control trials, indicating behavioral inhibition to threat. Behavioral inhibition was greatest amongst monkeys scored with most fearful temperament in the HIT. This primary result indicates the touch-screen task may be sensitive to a more subtle form of the bodily freeze behavior measured using the HIT. In Study 2, we tested whether these findings generalized to other classes of putatively negative stimuli; monkeys were shown pictures of the human intruder and objects associated with veterinary and husbandry procedures, interspersed with control trials (gray squares). There was no evidence of behavioral inhibition in Study 2. There was some evidence for response-slowing, which was greater for pictures of objects than pictures of the human intruder, and occurred independently of fearfulness in the HIT. We propose touch-screen tasks provide a more standardized and sensitive approach for assessing fearful temperament in laboratory primates.

Female Barbary macaque (Macaca sylvanus) copulation calls do not reveal the fertile phase but influence mating outcome.

In a number of primate species, females utter loud and distinctive calls during mating. Here we aim to clarify the information content and function of Barbary macaque (Macaca sylvanus) copulation calls by testing (i) whether or not copulation calls advertise the female fertile phase and (ii) whether and how copulation calls influence male ejaculatory behaviour. In order to do this, we combined hormone measurements with acoustic analysis and behavioural observations. In contrast to a previous study implying that the structure of copulation calls indicates the timing of the fertile phase, our results, using objective endocrine criteria for assessing ovulation, provide evidence that the structure of copulation calls of female Barbary macaques does not reveal the timing of the fertile phase. More importantly, females seem to influence the likelihood of ejaculation by calling versus remaining silent and by adjusting the timing of call onset. Females make use of this ability to influence mating outcome to ensure ejaculatory matings with almost all males in the group. In addition, calls given during ejaculatory copulations differ from those during non-ejaculatory copulations, providing information about mating outcome for listeners. We conclude that in this species, copulation calls apparently serve to enhance sperm competition and maximize paternity confusion.

Assessment of stress responses in rhesus macaques (Macaca mulatta) to daily routine procedures in system neuroscience based on salivary cortisol concentrations.

Non-human primates participating in neurophysiological research are exposed to potentially stressful experimental procedures, such as dietary control protocols, surgical implants and their maintenance, or social separation during training and experimental session. Here, we investigated the effect of controlled access to fluid, surgical implants, implant-related cleaning of skin margins, and behavioral training sessions on salivary cortisol levels of adult male rhesus macaques participating in neurophysiological research. The animals were trained to chew flavored cotton swabs to non-invasively collect saliva samples. Our data show no differences in cortisol levels between animals with and without implants, but both, controlled access to fluid and cleaning of implants individually increased salivary cortisol concentrations, while both together did not further increase the concentration. Specifically, before cleaning, individuals with controlled access to fluid had 55% higher cortisol concentrations than individuals with free access to fluid. Under free access to fluid, cortisol concentrations were 27% higher after cleaning while no effect of cleaning was found for individuals under controlled fluid access. Training sessions under controlled access to fluid also did not affect salivary cortisol concentrations. The observed changes in cortisol concentrations represent mild stress responses, as they are only a fraction of the range of the regular circadian changes in cortisol levels in rhesus monkeys. They also indicate that combinations of procedures do not necessarily lead to cumulative stress responses. Our results indicate that salivary cortisol levels of rhesus monkeys respond to neurophysiological experimental procedures and, hence, may be used to assess further refinements of such experimental methods.

Do acoustic features of lion, Panthera leo, roars reflect sex and male condition?

Long distance calls function to regulate intergroup spacing, attract mating partners, and/or repel competitors. Therefore, they may not only provide information about the sex (if both sexes are calling) but also about the condition of the caller. This paper provides a description of the acoustic features of roars recorded from 18 male and 6 female lions (Panthera leo) living in the Serengeti National park, Tanzania. After analyzing whether these roars differ between the sexes, tests whether male roars may function as indicators of their fighting ability or condition were conducted. Therefore, call characteristics were tested for relation to anatomical features as size, mane color, or mane length. Call characteristics included acoustic parameters that previously had been implied as indicators of size and fighting ability, e.g., call length, fundamental frequency, and peak frequency. The analysis revealed differences in relation to sex, which were entirely explained by variation in body size. No evidence that acoustic variables were related to male condition was found, indicating that sexual selection might only be a weak force modulating the lion's roar. Instead, lion roars may have mainly been selected to effectively advertise territorial boundaries.

Does the Structure of Female Rhesus Macaque Coo Calls Reflect Relatedness and/or Familiarity?

In social animals, kin relations strongly shape the social structure of a group. In female-bonded species, maternal relatedness is likely to be mediated via familiarity, but evidence is accumulating that non-human primates are able to recognize kin that they are not familiar with and adjust their behavior accordingly. In playback experiments, female rhesus macaques showed increased interest in 'coo' calls produced by unfamiliar paternal half-sisters compared to 'coo' calls produced by unfamiliar unrelated females, suggesting that these calls should have some common structural characteristics that facilitate the discrimination of kin from non-kin. Here we analyzed 'coo' calls of 67 adult female rhesus macaques from four groups and seven matrilines living on the island of Cayo Santiago (Puerto Rico). We tested whether the call structure of closely maternal and/or paternal related females, as determined from extensive pedigree data, differed from the call structure of unrelated females, while controlling for familiarity (i.e., group-matrilineal membership and age difference) of subjects. In contrast to our expectation, kinship did not predict similarities in 'coo' call structure, whereas 'coo' structure was more similar when produced by females of similar age as well as by females with higher familiarity, suggesting that experience is more decisive than genetic background. The high number of individuals in the analysis and the high accuracy of the assignment of calls to individuals render a lack of power as an unlikely explanation. Thus, based on the results of this study, kin recognition in rhesus monkeys does neither appear to be based on the assessment of self-similarity, nor on the comparison among related subjects (i.e., acoustic phenotype matching), but appears to be mediated by different or multiple cues. Furthermore, the results support the notion that frequent social interactions result in increasing acoustic similarity within largely innate call types ('vocal accommodation').

Male rhesus macaques use vocalizations to distinguish female maternal, but not paternal, kin from non-kin.

Recognizing close kin and adjusting one's behavior accordingly (i.e., favor kin in social interactions, but avoid mating with them) would be an important skill that can increase an animals' inclusive fitness. Previous studies showed that philopatric female rhesus macaques (Macaca mulatta) bias their social behavior toward maternal and paternal kin. Benefits gained from selecting kin should, however, not only apply to the philopatric sex, for which the enduring spatial proximity facilitates kin discrimination. Given that dispersal is costly, the dispersing sex may benefit from migrating together with their kin or into groups containing kin. In male rhesus macaques, natal migrants bias their spatial proximity toward familiar male kin rather than familiar non-kin. Here, we set up playback experiments to test if males use the acoustic modality to discriminate familiar female kin from non-kin in a non-sexual context. Males responded differently to the presentation of "coo" calls of related and unrelated females, with their reaction depending on the interaction between kin-line (maternal vs paternal kin) and degree of relatedness (r = 0.5, 0.25). Specifically, males were more likely to respond to close kin compared to more distant kin or unrelated females, with this effect being significant in the maternal, but not paternal kin-line. The present study adds to our knowledge of kin recognition abilities of the dispersing sex, suggesting that male rhesus macaques are also able to identify kin using the acoustic modality. We discuss that the probability of response might be affected by the potential benefit of the social partner.

Monkeys spontaneously discriminate their unfamiliar paternal kin under natural conditions using facial cues.

Kin recognition can enhance inclusive fitness via nepotism and optimal outbreeding. Mechanisms allowing recognition of patrilineal relatives are of particular interest in species in which females mate promiscuously, leading to paternity uncertainty. Humans are known to detect facial similarities between kin in the faces of third parties, and there is some evidence for continuity of this ability in nonhuman primates . However, no study has yet shown that this propensity translates into an ability to detect one's own relatives, one of the key prerequisites for gaining fitness benefits. Here we report a field experiment demonstrating that free-ranging rhesus macaques (Macaca mulatta) spontaneously discriminate between facial images of their paternal half-siblings and unrelated individuals, when both animals are unfamiliar to the tested individual. Specifically, subjects systematically biased their inspection time toward nonkin when the animals pictured were of their own sex (potential threats), relative to when they were of the opposite sex (potential mates). Our results provide strong evidence for visual phenotype matching and the first demonstration in any primate that individuals can spontaneously detect their own paternal relatives on the basis of facial cues under natural conditions.

Signaling in multiple modalities in male rhesus macaques: sex skin coloration and barks in relation to androgen levels, social status, and mating behavior.

The past decade has seen an increasing shift in animal communication towards more studies that incorporate aspects of signaling in multiple modalities. Although nonhuman primates are an excellent group for studying the extent to which different aspects of condition may be signaled in different modalities, and how such information may be integrated during mate choice, very few studies of primate species have incorporated such analyses. Here, we present data from free-ranging male rhesus macaques on sex skin coloration (modeled to receiver perception), bark vocal signals, androgen levels, morphometric variables, dominance status, and female mate choice. We show that, consistent with data on females, most intra- and interindividual variation in sex skin appearance occurs in luminance rather than color. Sex skin luminance was significantly correlated across different skin regions. Sex skin luminance did not correlate with the majority of bark parameters, suggesting the potential for the two signals to convey different information. Sex skin appearance was not related to androgen levels although we found some evidence for links between androgen levels and bark parameters, several of which were also related to morphometric variables. We found no evidence that either signal was related to male dominance rank or used in female mate choice, though more direct measures of female proceptive behavior are needed. Overall, the function of male sex skin coloration in this species remains unclear. Our study is among the first nonhuman primate studies to incorporate measurements of multiple signals in multiple modalities, and we encourage other authors to incorporate such analyses into their work.

Estrogen and Progestogen Correlates of the Structure of Female Copulation Calls in Semi-Free-Ranging Barbary Macaques (Macaca sylvanus).

Females of many Old World primates produce conspicuous vocalizations in combination with copulations. Indirect evidence exists that in Barbary macaques (Macaca sylvanus), the structure of these copulation calls is related to changes in reproductive hormone levels. However, the structure of these calls does not vary significantly around the timing of ovulation when estrogen and progestogen levels show marked changes. We here aimed to clarify this paradox by investigating how the steroid hormones estrogen and progesterone are related to changes in the acoustic structure of copulation calls. We collected data on semi-free-ranging Barbary macaques in Gibraltar and at La Forêt des Singes in Rocamadour, France. We determined estrogen and progestogen concentrations from fecal samples and combined them with a fine-grained structural analysis of female copulation calls (N = 775 calls of 11 females). Our analysis indicates a time lag of 3 d between changes in fecal hormone levels, adjusted for the excretion lag time, and in the acoustic structure of copulation calls. Specifically, we found that estrogen increased the duration and frequency of the calls, whereas progestogen had an antagonistic effect. Importantly, however, variation in acoustic variables did not track short-term changes such as the peak in estrogen occurring around the timing of ovulation. Taken together, our results help to explain why female Barbary macaque copulation calls are related to changes in hormone levels but fail to indicate the fertile phase.

Female ovarian cycle phase affects the timing of male sexual activity in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar.

Although all macaques have a multimale multifemale mating system, the degree of promiscuity shown by the Barbary macaque is considered to be extreme in terms of both mating frequency and number of mating partners. How mating activity is distributed throughout the female menstrual cycle and whether or not copulations are concentrated around the fertile phase as in other members of the genus is, however, not known. To examine this, we collected data on rates of copulation throughout 29 ovarian cycles from 13 free-ranging females of the Gibraltar Barbary macaque population and related them to the time of ovulation and the female fertile phase as determined from fecal hormone analysis. In addition, patterns of male inspection of females and time spent in consortship, both indicators of female attractivity, were also analyzed. The results indicate that both mating behavior and female attractivity vary predictably with ovarian cycle stage. Rates of copulation were found to increase toward the time of ovulation, with a distinct peak of ejaculatory (but not non-ejaculatory) copulations occurring in the fertile phase. Additionally, we show that frequency of inspection of females by males and time spent in consortship were also highest during the fertile phase and that ejaculatory copulations and male pericopulatory behaviors were significantly correlated with levels of female sex hormones. Our findings indicate that the Barbary macaque shows a mating pattern during the cycle similar to that described for other members of the genus. More importantly, however, our study provides clear evidence that despite an extreme degree of promiscuity Barbary macaque males concentrate their reproductive effort to the fertile phase, implying that they are able to discern this period and that thus timing of ovulation is not concealed from them. Estrogen-related cues appear to be involved in the process of recognition of female reproductive status by males, but the exact nature of these cues and how male Barbary macaques use them remains to be clarified.

Female sexual behavior and sexual swelling size as potential cues for males to discern the female fertile phase in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar.

Although female catarrhine primates show cyclic changes in sexual behavior and sexual swellings, the value of these sexual signals in providing information to males about timing of the fertile phase is largely unclear. Recently, we have shown that in Barbary macaques, males receive information from females which enables them to discern the fertile phase and to focus their reproductive effort accordingly. Here, we investigate the nature of the cues being used by examining female sexual behavior and the size of sexual swelling as potential indicators of the fertile phase. We collected behavioral data and quantified swelling size using digital images of 11 females of the Gibraltar Barbary macaque population and related the data to the time of ovulation and the fertile phase as determined from fecal hormone analysis. We found that rates of female sexual behaviors were not correlated with female estrogen levels and did not significantly differ between the fertile and non-fertile phases of the cycle. In contrast, swelling size was significantly correlated with female estrogen levels and increased predictably towards ovulation with size being maximal during the fertile phase. Moreover, frequencies of male ejaculatory copulations showed a strong positive correlation with swelling size and highest rates were found during maximum swelling. Our data provide strong evidence that female Barbary macaques honestly signal the probability of fertility through sexual swelling and that males apparently use this information to time their mating activities. Honest advertising of the fertile phase might be part of a female strategy to manipulate male mating behavior for their own advantage, such as ensure fertilization with high quality sperm or influence paternity outcome.