Affect Fertilization Success Research Articles

Differences in sperm morphology between Alouatta palliata and Alouatta pigra are consistent with the intensity of sperm competition in each species.

The intensity of sperm competition, in which sperm compete within the female reproductive tract to reach and fertilize her eggs, varies in species with different mating systems. Sperm competition is more intense in species where males cannot monopolize access to reproductive females and females mate with multiple males. In this scenario, a morphological change that increases the ability of sperm to reach and fertilize eggs should rapidly spread in the population, leading to sperm morphological differences between closely related species. Differences in sperm morphology have been reported among primate species with different mating systems. However, due to the inherent logistical and ethical difficulties to sample sperm from wild primates, the extent of variation in sperm morphology within species and among closely related species remains understudied. Here, we compared sperm morphological traits from two sister howler monkey species (Alouatta palliata and Alouatta pigra) that have different mating systems to investigate the effect of sperm competition on sperm morphological traits. We predicted that sperm from A. palliata, where females have more opportunities to mate with multiple males, would show differences in traits associated with increase sperm competitiveness compared to A. pigra where females mostly mate with the central male. We used linear mixed models to determine species differences in sperm morphology, controlling for individual variation. We found that midpieces and heads in A. palliata sperm were on average 26.2% and 11.0% longer, respectively, than those of A. pigra. Differences in these traits are important for sperm speed and hydrodynamic movement in other species and can affect fertilization success. This study provides empirical evidence of sperm morphological traits that evolved through sexual selection in sister primate species with different mating systems.

Constrained sex allocation after mating in a haplodiploid thrips species depends on maternal condition.

In females of haplodiploid animals, female production requires fertilization, whereas male production does not. Therefore, haplodiploid species can display extraordinary sex ratios. Constrained sex allocation occurs when a female cannot produce daughters. This can be due to virginity but may also occur after mating due to insemination failure, selfish genetic elements or physiological constraints. Here, we investigated the mechanism underlying constrained sex allocation in Pezothrips kellyanus. In this species some mated females produce highly female-biased broods, yet, for unknown reasons, others produce extremely male-biased broods. Using crossing experiments controlled for maternally inherited endosymbionts we confirmed that constrained females were successfully inseminated. Furthermore, male-biased offspring production was not paternally inherited, ruling out paternal sex ratio elements previously identified as sex ratio distorters in some parasitoid wasps. Next, we excluded mating time and paternal fitness effects (male size) on sex allocation. However, we found that constrained sex allocation only occurred in small females producing smaller eggs than large females producing larger eggs and female-biased broods. Consequently, the bimodal sex allocation pattern correlates with maternal condition, and may have arisen (adaptively or non-adaptively) in association with an egg size-mediated fertilization mechanism recently detected in some haplodiploids, with egg size positively affecting fertilization success.

Within-population sperm competition intensity does not predict asymmetry in conpopulation sperm precedence.

Postcopulatory sexual selection can generate evolutionary arms races between the sexes resulting in the rapid coevolution of reproductive phenotypes. As traits affecting fertilization success diverge between populations, postmating prezygotic (PMPZ) barriers to gene flow may evolve. Conspecific sperm precedence is a form of PMPZ isolation thought to evolve early during speciation yet has mostly been studied between species. Here, we show conpopulation sperm precedence (CpSP) between Drosophila montana populations. Using Pool-seq genomic data we estimate divergence times and ask whether PMPZ isolation evolved in the face of gene flow. We find models incorporating gene flow fit the data best indicating populations experienced considerable gene flow during divergence. We find CpSP is asymmetric and mirrors asymmetry in non-competitive PMPZ isolation, suggesting these phenomena have a shared mechanism. However, we show asymmetry is unrelated to the strength of postcopulatory sexual selection acting within populations. We tested whether overlapping foreign and coevolved ejaculates within the female reproductive tract altered fertilization success but found no effect. Our results show that neither time since divergence nor sperm competitiveness predicts the strength of PMPZ isolation. We suggest that instead cryptic female choice or mutation-order divergence may drive divergence of postcopulatory phenotypes resulting in PMPZ isolation.This article is part of the theme issue ‘Fifty years of sperm competition’.

Adult exposure to acidified seawater influences sperm physiology in Mytilus galloprovincialis: Laboratory and in situ transplant experiments

The ongoing increase of CO2 in the atmosphere is inducing a progressive lowering of marine water pH that is predicted to decrease to 7.8 by the end of this century. In marine environment, physical perturbation may affect reproduction, which is crucial for species’ survival and strictly depends on gamete quality. The effects of seawater acidification (SWAc) on gamete quality of broadcast spawning marine invertebrates result largely from experiments of gamete exposure while the SWAc impact in response to adult exposure is poorly investigated. Performing microcosm and in field experiments at a naturally acidified site, we investigated the effects of adult SWAc exposure on sperm quality parameters underlying fertilization in Mytilus galloprovincialis. These animals were exposed to pH 7.8 over 21 days and collected at different times to analyze sperm parameters as concentration, motility, viability, morphology, oxidative status, intra- and extra-cellular pH and mitochondrial membrane potential. Results obtained in the two experimental approaches were slightly different. Under field conditions, we found an increase in total sperm motility and mitochondrial membrane potential on days 7 and 14 from the start of SWAc exposure whereas, in microcosm, SWAc group showed an increase of total motility on day 14. In addition, sperm morphology and intracellular pH were affected in both experimental approaches; whereas oxidative stress was detected only in spermatozoa collected from mussels under natural SWAc. The overall analysis suggests that, in mussels, SWAc toxic mechanism in spermatozoa does not involve oxidative stress. This study represents the first report on mussel sperm quality impairment after adult SWAc exposure, which may affect fertilization success with negative ecological and economic consequences; it also indicates that, although naturally acidified areas represent ideal natural laboratories for investigating the impact of ocean acidification, microcosm experiments are necessary for examining action mechanisms.

Measuring sperm swimming performance in birds: effects of dilution, suspension medium, mechanical agitation, and sperm number

Sperm swimming performance, including swimming speed and the proportion of motile cells, may strongly affect fertilization success. However, little is known about how methodological factors affect in vitro measurement of these parameters. We compare the swimming performance of sperm from House Sparrows (Passer domesticus) and Spanish Sparrows (Passer hispaniolensis) in two standard suspension media, at two different dilutions, and with different degrees of cell agitation. Further, we conduct a resampling analysis to investigate sample size effects. Sperm performance was generally reduced when sperm were diluted, or when suspended in phosphate-buffered saline (PBS) rather than a medium containing additional nutrients. Sperm performance was particularly low when they were diluted more and suspended in PBS, suggesting that seminal fluid may provide compounds that enhance performance but that these are less available following dilution. Mechanical agitation of the cells by vigorous pipetting increased the proportion of motile cells. Between-male repeatability, assessed on a single sample measured in multiple conditions, was moderate and significant, suggesting that similar results may be obtained regardless of the methodology used to assess sperm motion. We found no evidence of biased results when low numbers of cells per male were used in analysis, though precision increased substantially as sample size increased from five to 20 or more cells per male. We recommend using the same suspension media and similar sperm concentrations and levels of agitation, to the greatest degree possible, and including as many individuals as possible in analyses, even when some individuals are represented by few sperm cells.

Swimming kinematics and temperature effects on spermatozoa from wild and captive shortnose sturgeon (Acipenser brevirostrum)

Computer-assisted semen analysis (CASA) and cluster analysis were used to compare spermatozoa swimming kinematics and milt quality between wild and captive shortnose sturgeon (Acipenser brevirostrum). Milt samples from 27 shortnose sturgeon were collected in May 2016 and June 2017. Of these, 19 were wild caught in the Saint John River, New Brunswick, Canada, and eight were from a captive population at the Mactaquac Biodiversity facility. The following kinematic variables were measured immediately following sperm activation (˜5 s), at 30, 60, and 180 s post-activation; average path velocity (VAP); straight-line velocity (VSL); curvilinear velocity (VCL); amplitude of lateral head displacement (ALH); beat cross frequency (BCF); straightness (STR); linearity (LIN); wobble (WOB); percent motility (MOT). Analyses were conducted at 7, 10, and 14 °C to determine potential effects of temperature on kinematics. Principal components analysis (PCA) of original kinematic variables yielded two main components, a speed/wobble component along with a movement pattern component. Hierarchical cluster analysis (HCPC) indicated there were distinct subpopulations, with composition of clusters the result of fish source (wild-caught or captive). Wild-caught fish had greater sperm densities (P = 0.0064) and sperm swimming speeds compared to captive fish (P < 0.05). Temperature had a significant effect only on captive spermatozoa, and this result was not consistent between time periods. There was no effect of hormonal manipulation on spermatozoa motility kinematics. Results indicate there are significant differences in measures of milt quality between wild and captive shortnose sturgeon, indicating an effect of rearing condition on reproductive potential, which may affect fertilization success.

Role of Sex Ratio and Density on Fertilization Success of Intensively Cultured Endangered Woundfin

Abstract Woundfin Plagopterus argentissimus are a small, endangered cyprinid found solely in the Virgin River across Utah, Arizona, and Nevada. As of now, only three facilities culture Woundfin for conservation propagation purposes, but there is growing concern that current extensive culture protocols are not meeting the conservation recovery goals outlined in the Virgin River Resource Management and Recovery Program. The development of alternative rearing methodology, like effective intensive culture conditions, could help increase production to more effectively meet the conservation propagation needs for this species. Our goal was to assess the effect of different sex ratios and fish densities on reproductive success at indoor facilities. We measured fertilization success at three different sex ratios (1:1, 1:3, 1:5; female:male) and three different fish densities (0.05, 0.21, and 0.47 fish/L) in a laboratory setting. Sex ratio did not significantly affect fertilization success (P = 0.73). Fertilization was completely inhibited at a density of 0.05 fish/L, but there was no difference in fertilization success between the two other density treatments (P = 0.92). Our findings suggest that a density greater than 0.05 fish/L and a sex ratio of 1:1, 1:3, or 1:5 will likely enhance fertilization success. The results and conclusions of this paper provide valuable rearing information for the Virgin River Resource Management and Recovery Program and hatchery managers attempting to increase production of Woundfin in an intensive culture setting.

Sperm competition and the evolution of precopulatory weapons: Testis size and amplexus position, but not arm strength, affect fertilization success in a chorusing frog.

Trade-offs between pre- and postcopulatory traits influence their evolution, and male expenditure on such traits is predicted to depend on the number of competitors, the benefits from investing in weapons, and the risk and intensity of sperm competition. Males of the chorusing frog Crinia georgiana use their arms as weapons in contest competition. Previously, we showed that increased numbers of rivals elevated the risk and intensity of sperm competition due to multimale amplexus, and caused a reversal in the direction of precopulatory selection on arm girth. Here, we focused on the factors affecting postcopulatory fertilization success during group spawning, using paternity data from natural choruses. Competitive fertilization success depended on the time spent amplexed and amplexus position. Relative testes size but not arm girth, contributed to fertilization success, but the effect of testes size depended on amplexus position. Our findings offer within species empirical support for recent sperm competition models that incorporate precopulatory male-male competition, and show why an understanding of the evolution of animal weapons requires a consideration of both pre- and postcopulatory episodes of sexual selection.

Pollen heteromorphism is pervasive in Thalictrum (Ranunculaceae)

Among the angiosperms, features of pollen morphology such as grain size, aperture number and surface ornamentation display striking variation. It is less well appreciated that pollen morphology may vary within and among populations of the same species as well as within individual plants. In some species, individual plants produce multiple types of fertile pollen grains (called pollen heteromorphism). Aspects of pollen morphology, such as aperture number, are likely to affect fertilization success with different morphologies favored in different local competitive and ecological environments. This study surveys variation in pollen grain morphology among species throughout the genus Thalictrum. Pollen from individuals of 36 species was rehydrated from herbarium specimens, and light microscopy was used to quantify pollen grain aperture number and size. I find that pollen aperture-number heteromorphism is present within all Thalictrum species studied, and distributions of aperture-number morphs vary both within and among species. This study provides an example of significant pollen heteromorphism within a genus that also varies widely for pollination mode and sexual system.

Long sperm fertilize more eggs in a bird.

Sperm competition, in which the ejaculates of multiple males compete to fertilize a female's ova, results in strong selection on sperm traits. Although sperm size and swimming velocity are known to independently affect fertilization success in certain species, exploring the relationship between sperm length, swimming velocity and fertilization success still remains a challenge. Here, we use the zebra finch (Taeniopygia guttata), where sperm size influences sperm swimming velocity, to determine the effect of sperm total length on fertilization success. Sperm competition experiments, in which pairs of males whose sperm differed only in length and swimming speed, revealed that males producing long sperm were more successful in terms of (i) the number of sperm reaching the ova and (ii) fertilizing those ova. Our results reveal that although sperm length is the main factor determining the outcome of sperm competition, complex interactions between male and female reproductive traits may also be important. The mechanisms underlying these interactions are poorly understood, but we suggest that differences in sperm storage and utilization by females may contribute to the outcome of sperm competition.

Ask not (only) what proteomics can do for behavior, but (also) what behavior can do for proteomes: a comment on Valcu and Kempenaers

Valcu and Kempenaers’ (2015) paper on the use of proteomics in behavioral ecology is a tour de force detailing everything from how different proteomics methods work and how to choose among them, to the ever-increasing use of proteomics to answer and/ or generate questions in behavioral ecology, to a roadmap for future intersections between proteomics and behavioral ecology. Their insights should be of great value to researchers interested in this area of study. In addition to the view that proteomics can contribute to the study of behavior, the study of behavior will contribute to our understanding of proteomes, and how those are modulated with condition and time. In this commentary, I illustrate this view with examples from studies of seminal fluid proteins (SFPs). SFPs are transferred by males to females during mating and, in some species, can influence female behavior and physiology in ways that affect the reproductive success of both partners. Several studies have recently used proteomics to determine which proteins are transferred from males to females in the seminal fluid or as part of the sperm (e.g., Findlay et al. 2008). Studies of specific proteins have revealed how these proteins affect fertilization success and female postmating responses (e.g., Rogers et al. 2009). These studies have also uncovered situations in which behavior can shape the proteome.

Species and gamete-specific fertilization success of two sea urchins under near future levels of pCO2

Since the Industrial Revolution, rising atmospheric CO2 concentration has driven an increase in the partial pressure of CO2 in seawater (pCO2), thus lowering ocean pH. We examined the separate effects of exposure of gametes to elevated pCO2 and low pH on fertilization success of the sea urchin Strongylocentrotus nudus. Sperm and eggs were independently exposed to seawater with pCO2 levels ranging from 380 (pH7.96–8.3) to 6000ppmv (pH7.15–7.20). When sperm were exposed, fertilization rate decreased drastically with increased pCO2, even at a concentration of 450ppmv (pH range: 7.94 to 7.96). Conversely, fertilization of Hemicentrotus pulcherrimus was not significantly changed even when sperm was exposed to pCO2 concentrations as high as 750ppmv. Exposure of S. nudus eggs to seawater with high pCO2 did not affect fertilization success, suggesting that the effect of increased pCO2 on sperm is responsible for reduced fertilization success. Surprisingly, this result was not related to sperm motility, which was insensitive to pCO2. When seawater was acidified using HCl, leaving pCO2 constant, fertilization success in S. nudus remained high (>80%) until pH decreased to 7.3. While further studies are required to elucidate the physiological mechanism by which elevated pCO2 impairs sperm and reduces S. nudus fertilization, this study suggests that in the foreseeable future, sea urchin survival may be threatened due to lower fertilization success driven by elevated pCO2 rather than by decreased pH in seawater.

Wild Atlantic cod sperm motility is negatively affected by ovarian fluid of farmed females

Atlantic cod escape from fish farms at higher rates than commonly cultured marine species, and escapees have been observed to interact with wild fish in mating aggregations. Pre- vious research suggests that potential interbreeding is mediated largely by the likelihood of wild males spawning with escaped females, and as such, the egg and ovarian fluid characteristics of these females could affect fertilization success and the likelihood of hybridization. Farmed cod have been noted to have poor egg quality compared to wild individuals, and some of this differ- ence may be due to the ovarian fluid, which can affect key sperm-motility parameters related to fertilization success. We tested the hypothesis that the ovarian fluid of farmed females negatively affects the sperm performance of wild males. Sperm-motility parameters and fertilization capacity of wild male sperm were analyzed in the presence of both farmed and wild female ovarian fluid. Sperm performed similarly in the presence of wild female ovarian fluid and a seawater control. Ovarian fluid of farmed females negatively affected sperm swimming and the capacity to fertilize eggs. These differences may be related to nutritional deficiencies of farmed individuals. Although it has been demonstrated that wild males actively court farmed females, our results indicate that their ovarian fluid quality can inhibit fertilization success.

Tactic-specific differences in seminal fluid influence sperm performance

Seminal fluid often makes up a large part of an ejaculate, yet most empirical and theoretical studies on sperm competition have focused on how sperm characteristics (number and quality) affect fertilization success. However, seminal fluid influences own sperm performance and may potentially influence the outcome of sperm competition, by also affecting that of rivals. As a consequence males may be expected to allocate their investment in both sperm and seminal fluid in relation to the potential level of competition. Grass goby (Zosterisessor ophiocephalus) is an external fertilizer with guard-sneaker mating tactics, where sperm competition risk varies according to the tactic adopted. Here, we experimentally manipulated grass goby ejaculates by separately combining sperm and seminal fluid from territorial and sneaker males. While sperm of sneaker and territorial males did not differ in their performance when they interacted with their own seminal fluid only, sperm of sneakers increased their velocity and fertilization rate in the presence of territorial males' seminal fluid. By contrast, sneaker males' seminal fluid had a detrimental effect on the performance of territorial males' sperm. Sperm velocity was unaffected by the seminal fluid of males employing the same tactic, suggesting that seminal fluid's effect on rival-tactic sperm is not based on a self/non-self recognition mechanism. Our findings show that cross interactions of sperm and seminal fluid may influence the fertilization success of competing ejaculates with males investing in both sperm and seminal fluid in response to sperm competition risk.

Sperm motility of the scleractinian coral Acropora digitifera under preindustrial, current, and predicted ocean acidification regimes

Ocean acidification caused by the uptake of anthropogenic CO2 in the oceans negatively affects the early life stages of corals by reducing their calcification rate. Acidification also inhibits the sperm motility of corals, potentially affecting fertilization success. We investi- gated the effects of different pCO2 (partial pressure of CO2) conditions on the sperm motility of Acropora digitifera. Using a pCO2-control system, we maintained pCO2 at concentrations from preindustrial and present-day levels up to the level predicted by the year 2100 (300, 400, and 1000 ppm, respectively). Our results indicated that ocean acidification has the potential to sup- press the sperm flagellar motility of A. digitifera. Furthermore, sperm motility will likely decline by ~30%, which may impact fertility, if the sensitivity of sperm motility to decreasing pH cannot adapt over a span of ~90 yr.

Males responding to sperm competition cues have higher fertilization success in a soldier fly

Sperm competition has been demonstrated to be an important force shaping male behavior in a number of species. For example, males may prolong copulation duration when they perceive sperm competition to be high. Although male behavioral responses to sperm competition have been shown in several species, their effects on reproductive success have rarely been demonstrated. In the soldier fly Merosargus cingulatus, males prolong copulations when sperm competition is high and when mating with more fecund females. Here, I tested the hypothesis that this behavioral response results in higher reproductive success for males. I exposed males to different simulated levels of sperm competition (high or low male density at the oviposition site) then introduced a female. I allowed the pair to mate and the female to oviposit. I determined the percentage of offspring sired by the male using amplified fragment length polymorphism profiles. Sperm competition did not affect clutch size, but it did affect fertilization success: males under higher simulated sperm competition increased copulation duration and fertilized a higher percentage of a female's egg clutch than did males under lower sperm competition.

Effects of timing of nest entry and body size on the fertilization success of alternative male reproductive phenotypes of masu salmon (Oncorhynchus masou)

Using a natural population of masu salmon ( Oncorhynchus masou (Brevoort, 1856)) in a stream of Shikaribetsu Lake, Hokkaido, Japan, we combined behavioural observations with genetic parentage analysis to explore the factors affecting fertilization success achieved by alternative mating tactics (fighting by large migratory males and sneaking by small mature male parr). Larger males gained priority access to females; migrant males do this by holding a guarding position near the nesting female and mature male parr do this by adopting sneaker behaviour and attending spawning groups. Status of mature male parr was related to success of nest entry but not to timing of nest entry, although the timing of nest entry influenced fertilization success of sneakers and ejaculation simultaneous with pair spawning was needed for fertilization by sneakers. The relative body size of each male who successfully spawned with a female is also likely to determine the proportion of eggs he fertilized because larger males have larger ejaculate. These results provide insight into factors relating to variation in fertilization success, how body size dimorphisms may be related to fitness, and evolution of alternative mating tactics.

Male and female effects on fertilization success and offspring viability in the Peron's tree frog,Litoria peronii

Abstract There is increasing theoretical and empirical evidence that genetic compatibility among partners is an important determinant of fertilization success and offspring viability. In amphibians, females often actively choose partners from among a variety of males and polyandry is common. Genetic compatibility among partners may therefore be an important determinant of fertilization success and offspring viability in some amphibians. Amphibians also show some of the highest levels of genetic differentiation among neighbouring populations known in vertebrates, and as such, populations may have evolved different co‐adapted gene complexes. This means that offspring from among‐population crosses may have reduced fitness. It is therefore essential to understand to what extent crossings between and within populations may interfere with successful fertilization and offspring viability. Here, we test whether crossing individuals within and between two different populations of the Australian Peron's tree frog (Litoria peronii) using artificial fertilizations affect fertilization success and offspring viability. Fertilization success per se is strongly influenced by male identity, which is likely to depend at least to some extent on the experimental procedure (e.g. resulting in variation in sperm number per ejaculate), whereas there was no fertilization effect of female identity. More importantly, male and female identity, independently of each other, explained significant variation in offspring viability, whereas no such effect could be linked to population of origin. Thus, our experiments suggest that crossing populations may not always be the most significant factor affecting fertilization success or offspring viability, but may be more influenced by the genetic quality or the genetic compatibility of partners.

Fertilization success in &lt;i&gt;Galeolaria caespitosa&lt;/i&gt; (Polychaeta: Serpillidae): gamete characteristics, role of sperm dilution, gamete age, and contact time

Variability in gamete traits and the factors affecting fertilization success were studied in a common gregarious broadcast-spawning serpulid polychaete Galeolaria caespitosa. Variability of egg size and sperm velocity were not related to the adult size. High sperm concentrations (107-108 sperm. ml-1) were required to achieve fertilization rates of 60-80%. The gamete contact time required to achieve high fertilization rates (70-80%) in G. caespitosa did not exceed 5 minutes of gamete exposure given sufficiently high sperm concentrations. This suggests that attachment of sperm to the egg takes place very rapidly, and a short-term exposure to highly concentrated sperm is a common feature of fertilization ecology of this gregarious species. A sharp decline in fertilization rates at high sperm concentrations usually attributable to polyspermy was not observed. Sperm were motile for up to 6 hours after activation; however, swimming velocity and fertilization success decreased after 2 hours. Eggs of G. caespitosa were fertilizable up to 10 hours after spawning, but the number of embryos resulting from fertilizations by fresh sperm decreased after 2 hours. The gamete traits of G. caespitosa appear to have evolved to enable this sessile organism to reproduce under conditions of high population density and increased risk of polyspermy.

Effect of Photoperiod Manipulation on Broodstock Spawning, Fertilization Success, and Egg Developmental Abnormalities in Atlantic Cod, Gadus morhua

Abstract Spawning activity in two captive Atlantic cod broodstock groups previously captured from the wild stock fishery was monitored over three spawning seasons. Both groups spawned under ambient photoperiod (PP) in the first year, after which photomanipulation was applied to compress the PP cycle of one broodstock group, while the second group was maintained on natural PP. Increased total egg volumes, number of egg batches, total egg production, and duration of the spawning season were observed in both ambient PP and advanced PP groups in the second and third spawning seasons. Compared to ambient controls, the photoadvanced group commenced spawning earlier, had a longer spawning season, produced a lower total volume of eggs over the entire spawning season, and had lower mean daily batch volumes of eggs, and the eggs were of smaller mean diameter. Fertilization success was &gt;90% in all years, increased in both groups in the first year after photomanipulation but declined to prior levels in the following year. No consistent difference in fertilization success was observed between advanced PP and ambient PP groups. Rates of cell development abnormalities were low (&lt;10%) in both groups over the three spawning seasons. Abnormalities of cell symmetry and clarity increased in both ambient PP and advanced PP groups over the three spawning seasons. However, no consistent differences in rate of cell abnormalities were observed between advanced PP and ambient PP groups within years. We conclude that PP advancement is an effective technique to compress the time between successive spawning periods in Atlantic cod and does not negatively affect fertilization success or rates of cell developmental abnormalities. However, it has a negative effect on both volume and size of eggs produced.