Sex Ratio Manipulation Research Articles

Socially plastic responses in females are robust to evolutionary manipulations of adult sex ratio and adult nutrition.

Socially plastic behaviours are widespread among animals and can have a significant impact on fitness. Here, we investigated whether the socially plastic responses of female Drosophila melanogaster can evolve in predictable ways following long-term manipulation of adult sex ratio and adult nutrient availability. Previous reports show that female D. melanogaster respond plastically to their same-sex social environment and lay significantly fewer eggs after mating when previously exposed to other females. In this study, we tested 2 hypotheses, using females drawn from lines with an evolutionary history of exposure to variation in adult sex ratio (male-biased, female-biased or equal sex ratio) and adult nutritional environment (high or low quality). The first was that a history of elevated competition in female-biased regimes would select for increased plastic fecundity responses in comparison to females from other lines. The second was that these responses would also be magnified under poor nutritional resource regimes. Neither hypothesis was supported. Instead, we found that plastic fecundity responses were retained in females from all lines and did not differ significantly across any of them. The lack of differences does not appear to be due to insufficient selection, as we did observe significant evolutionary responses in virgin egg-laying patterns according to sex ratio and nutritional regime. The lack of variation in the magnitude of predicted plasticity is consistent with the idea that the costs of maintaining plasticity are low, benefits high, and that plasticity itself can be relatively hard wired.

Identifying the sex chromosome and sex determination genes in the cattle tick, Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is globally one of the most economically important ectoparasites of cattle costing the agriculture industry billions of dollars annually. Resistance to chemical control measures has prompted the development of novel methods of control. Recent advancements in genetic control measures for human and other animal vectors have utilized sex determination research to manipulate sex ratios, which have shown promising results in mosquitoes namely Aedes aegypti and Anopheles stephensi. Here, we use R. (B.) microplus as a model to provide foundational research to allow similar avenues of investigation in ticks using R. (B.) microplus as a model. Karyotypes for R. (B.) microplus show an XX:XO sex determining system with the largest chromosome being the sex chromosome. Using flow cytometric methods, the size of the sex chromosome was estimated at 526.91 Mb. All measures to identify the sex chromosome within the cattle tick genome assembly associated sex chromosomal characteristics to two chromosomes. This discrepancy between the assembly and karyotypes of the tick led to generating a new genome assembly with a single adult male specimen. The two chromosomes in question aligned with a single scaffold within the new genome that had a length of 513.29 Mb and was the first time the sex chromosome was identified in an Ixodid genome assembly.

A decade-long study demonstrates that a population of invasive sea lamprey (Petromyzon marinus) can be controlled by introducing sterilized males

The release of sterilized insects to control pest populations has been used successfully during the past 6 decades, but application of the method in vertebrates has largely been overlooked or met with failure. Here, we demonstrate for the first time in fish, that a small population of sea lamprey (Petromyzon marinus; Class Agnatha), arguably one of the most impactful invasive fish in the world, can be controlled by the release of sterilized males. Specifically, the release of high numbers of sterile males (~ 1000's) into a geographically isolated population of adult sea lamprey resulted in the first multiyear delay in pesticide treatment since treatments began during 1966. Estimates of percent reduction in recruitment of age-1 sea lamprey due to sterile male release ranged from 7 to 99.9% with the precision of the estimate being low because of substantial year-to-year variability in larval density and distribution. Additional monitoring that accounts for recruitment variability in time and space would reduce uncertainty in the degree to which sterile male release reduces recruitment rates. The results are relevant to vertebrate pest control programs worldwide, especially as technical opportunities to sterilize vertebrates and manipulate sex ratios expand.

A possible case of offspring sex manipulation as result of a biased adult sex ratio

Although random meiosis should prevent the facultative adjustment of offspring sex ratio, theory predicts that females should produce more of the sex with the higher reproductive value. We reported a case of offspring sex ratio manipulation in grass wrens Cistothorus platensis. Males in better body condition would have higher reproductive value than females due to the potential for social polygyny and extra-pair fertilizations. On the other hand, local demography influences reproductive strategies in grass wrens as male abundance affects both social polygyny and extra-pair paternity frequencies. We evaluated whether females bias their brood sex ratio in response to adult sex ratio and nestling body condition (a proxy for female’s prospects of producing high-quality males). Females raised more male offspring when males were less abundant in the population (female-biased adult sex ratio). However, we found no relationship between nestling body condition and brood sex ratio, suggesting that females did not bias the brood sex ratio towards males when able to raise nestlings in better body condition. Taken together, our results provide the first suggestive evidence that female birds can manipulate their offspring sex ratio in response to the adult sex ratio.

Genotypes and phenotypes in a Wolbachia-ant symbiosis.

The fitness effects of overt parasites, and host resistance to them, are well documented. Most symbionts, however, are more covert and their interactions with their hosts are less well understood. Wolbachia, an intracellular symbiont of insects, is particularly interesting because it is thought to be unaffected by the host immune response and to have fitness effects mostly focussed on sex ratio manipulation. Here, we use quantitative PCR to investigate whether host genotype affects Wolbachia infection density in the leaf-cutting ant Acromyrmex echinatior, and whether Wolbachia infection density may affect host morphology or caste determination. We found significant differences between host colonies in the density of Wolbachia infections, and also smaller intracolonial differences in infection density between host patrilines. However, the density of Wolbachia infections did not appear to affect the morphology of adult queens or likelihood of ants developing as queens. The results suggest that both host genotype and environment influence the host-Wolbachia relationship, but that Wolbachia infections carry little or no physiological effect on the development of larvae in this system.

On the Origin and Evolution of the Mosquito Male-determining Factor Nix.

The mosquito family Culicidae is divided into 2 subfamilies named the Culicinae and Anophelinae. Nix, the dominant male-determining factor, has only been found in the culicines Aedes aegypti and Aedes albopictus, 2 important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution of Nix. Except for the Culex genus, Nix homologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from 3 highly divergent tribes comprising 4 genera, suggesting Nix originated at least 133 to 165 million years ago (MYA). Heterologous expression of 1 of 3 divergent Nix open reading frames (ORFs) in Ae. aegypti resulted in partial masculinization of genetic females as evidenced by morphology and doublesex splicing. Phylogenetic analysis suggests Nix is related to femaleless (fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes. Nix from all species has a conserved structure, including 3 RNA-recognition motifs (RRMs), as does fle. However, Nix has evolved at a much faster rate than fle. The RRM3 of both Nix and fle are distantly related to the single RRM of a widely distributed and conserved splicing factor transformer-2 (tra2). The RRM3-based phylogenetic analysis suggests this domain in Nix and fle may have evolved from tra2 or a tra2-related gene in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex determination in mosquitoes and will inform broad applications of mosquito-control strategies based on manipulating sex ratios toward nonbiting males.

No evidence of sex ratio manipulation by black-throated blue warblers in response to food availability

No evidence of sex ratio manipulation by black-throated blue warblers in response to food availability

Adult sex ratios affect mating behaviour in the common housefly Musca domestica L. (Diptera; Muscidae)

Abstract Adult sex ratio determines the level of mate availability and intrasexual competition for each sex. Sex ratio biases have been proposed to enhance the productivity of animal rearing procedures. However, behaviour may change in response to sex ratio manipulations that may counteract potential benefits. We investigated how sex ratios affected mating behaviour of the housefly Musca domestica, a species used in the animal feed industry. We hypothesized a reduced courtship effort and mating latency and increased ejaculate allocation (copulation duration) under male-biased sex ratios, whereas female-biased sex ratios would lead to the opposite effects. However, courtship effort was reduced in female-biased groups, implying reduced male harassment. Mating latency was lower and copulation lasted longer in female-biased groups, which may reduce reproduction time and increase female fecundity and lifespan. Our results indicate that in houseflies, female-biased sex ratios cause behavioural changes in both sexes that could positively contribute to reproductive output.

The presence of a guard vicariously drives split sex ratios in a facultatively social bee.

Split sex ratios provide broad insights into how reproductive strategies evolve, and historically have special relevance to the evolution of eusociality. Yet almost no attention has been directed to situations where split sex ratios may potentially decrease the payoffs for worker-like behaviour, increasing selective thresholds for eusociality. We examined sex ratios in a facultatively social colletid bee, Amphylaeus morosus. Sex ratios in this bee vary strongly with the presence of a nest guard and in a pattern that does not conform to assumptions of previous models in which split sex ratios facilitate altruism. While the production of daughters was constant across social and solitary nests, mothers produced more brood when a non-reproductive guard was present, but these extra brood were all male. This leads to split sex ratios, vicariously driven by guards that are unable to manipulate sex ratios in their favour. Importantly, if guarding becomes more common in a population this would lead to an excess of males and lower the genetic value of these extra males to guards, effectively putting a brake on selection for worker-like behaviour.

Kangaroo Fathers Modulate Maternal Control of Offspring Sex but Not Postpartum Maternal Expenditure.

AbstractWhen sons and daughters have different fitness costs and benefits, selection may favor deviations from an even offspring sex ratio. Most theories on sex ratio manipulation focus on maternal strategies and sex-biased maternal expenditure. Recent studies report paternal influences on both offspring sex ratio and postpartum sex-biased maternal expenditure. We used long-term data on marked kangaroos to investigate whether and how paternal mass and skeletal size, both determinants of male reproductive success, influenced (a) offspring sex in interaction with maternal mass and (b) postpartum sex-biased maternal expenditure. When mothers were light, the probability of having a son increased with paternal mass. Heavy mothers showed the opposite trend. A similar result emerged when considering paternal size instead of mass. Postpartum maternal sex-specific expenditure was independent of paternal mass or size. Studies of offspring sex manipulation or maternal expenditure would benefit from an explicit consideration of paternal traits, as paternal and maternal effects can modulate each other.

Factors influencing dispersal initiation and timing in a facultative cooperative breeder

AbstractNatal dispersal is a high-risk endeavor where decisions on whether and when to disperse have long-term consequences. Among facultative cooperative breeders, juveniles often forego dispersal and remain philopatric for one or more breeding seasons. This decision is key to the formation of cooperative breeding groups and could have significant effects on reproductive success. We investigated the probability and initiation of dispersal in the cooperatively breeding Brown-headed Nuthatch (Sitta pusilla) to determine the influence that social environments had on dispersal. This study was concurrent with another study where manipulation of population sex ratios increased the prevalence and size of cooperative groups. The concurrent manipulations enabled us to evaluate social effects on dispersal as a plausible mechanism driving the relationship between adult sex ratios and cooperation. We evaluated which factors best predicted whether males dispersed, and the timing of dispersal for both sexes. We considered variables related to the immediate nesting environment as well as characteristics of the local population. Social environments were related to dispersal for both males and females. Juvenile males dispersed earlier when a helper was present in the natal group. Females dispersed earlier in settings with more adult neighbors and when a lower proportion of those neighbors were males. Females with shorter tarsi relative to their siblings dispersed earlier, suggesting that size-based competitive interactions may also affect dispersal decisions. Our results suggest juveniles disperse more readily when they fledge in constrained social environments, and that competition with conspecifics is a major driver of dispersal in the Brown-headed Nuthatch.

Sperm Limitation Produces Male Biased Offspring Sex Ratios in the Wasp, Nasonia vitripennis (Hymenoptera: Pteromalidae)

Haplo-diploid sex determination in the parasitoid wasp, Nasonia vitripennis (Walker), allows females to adjust their brood sex ratios. Females influence whether ova are fertilized, producing diploid females, or remain unfertilized, producing haploid males. Females appear to adjust their brood sex ratios to minimize ‘local mate competition,’ i.e., competition among sons for mates. Because mating occurs between siblings, females may optimize mating opportunities for their offspring by producing only enough sons to inseminate daughters when ovipositing alone, and producing more sons when superparasitism is likely. Although widely accepted, this hypothesis makes no assumptions about gamete limitation in either sex. Because sperm are used to produce daughters, repeated oviposition could reduce sperm supplies, causing females to produce more sons. In contrast, if egg-limited females produce smaller broods, they might use fewer sperm, making sperm limitation less likely. To investigate whether repeated oviposition and female fertility influence gamete limitation within females, we created two treatments of six mated female wasps, which each received a series of six hosts at intervals of 24 or 48 h. All females produced at least one mixed-sex brood (63 total broods; 3,696 offspring). As expected, if females became sperm-limited, in both treatments, brood sex ratios became increasingly male-biased with increasing host number. Interhost interval did not affect brood size, total offspring number, or sex ratio, indicating females did not become egg limited. Our results support earlier studies showing sperm depletion affects sex allocation in N. vitripennis¸ and could limit adaptive sex ratio manipulation in these parasitoid wasps.

Propagule size and sex ratio influence colonisation dynamics after introduction of a non-native lizard.

The composition of founding populations plays an important role in colonisation dynamics and can influence population growth during early stages of biological invasion. Specifically, founding populations with small propagules (i.e. low number of founders) are vulnerable to the Allee effect and have reduced likelihood of establishment compared to those with large propagules. The founding sex ratio can also impact establishment via its influence on mating success and offspring production. Our goal was to test the effects of propagule size and sex ratio on offspring production and annual population growth following introductions of a non-native lizard species (Anolis sagrei). We manipulated propagule composition on nine small islands, then examined offspring production, population growth and survival rate of founders and their descendants encompassing three generations. By the third reproductive season, per capita offspring production was higher on islands seeded with a relatively large propagule size, but population growth was not associated with propagule size. Propagule sex ratio did not affect offspring production, but populations with a female-biased propagule had positive growth, whereas those with a male-biased propagule had negative growth in the first year. Populations were not affected by propagule sex ratio in subsequent years, possibly due to rapid shifts towards balanced (or slightly female biased) population sex ratios. Overall, we show that different components of population fitness have different responses to propagule size and sex ratio in ways that could affect early stages of biological invasion. Despite these effects, the short life span and high fecundity of A. sagrei likely helped small populations to overcome Allee effects and enabled all populations to successfully establish. Our rare experimental manipulation of propagule size and sex ratio can inform predictions of colonisation dynamics in response to different compositions of founding populations, which is critical in the context of population ecology and invasion dynamics.

Determinants of offspring sex in kangaroos: a test of multiple hypotheses

Abstract When the fitness costs and benefits of sons and daughters differ, offspring sex ratio manipulation could be an important reproductive tactic. We explored the effects of environment and maternal caring ability on offspring sex to test four adaptive sex ratio modification hypotheses: the extrinsic modification hypothesis (EMH), carrying capacity hypothesis (CCH), Trivers-Willard hypothesis (TWH), and cost-of-reproduction hypothesis (CRH). The EMH and CCH propose that environmental conditions shape offspring sex ratios, directly or in interaction with maternal condition. The TWH and CRH predict a positive relationship between maternal condition and production of the costlier sex. The TWH predicts that mothers with superior caring ability should produce more of the sex that can provide the greatest fitness returns from additional maternal allocation, and the CRH proposes that females with limited caring ability should reduce fitness costs by producing the cheaper sex. Repeated measures on 83 known-age eastern gray kangaroos, polygynous marsupials with strong sexual dimorphism, revealed that offspring sex ratio was independent of per capita forage, supporting neither the EMH nor CCH, but was dependent on maternal mass, consistent with the TWH and CCH. Our results, however, cannot clearly identify the ultimate cause of the relationship between maternal mass and greater production of sons. One of the three assumptions of the TWH could not be verified, and mothers of sons suffered only marginal additional fitness costs. Sex ratios in higher vertebrates are likely not solely explained by factors dependent on maternal control.

Exogenous Progesterone for Offspring Sex Ratio Manipulation in Arabic Chickens (Gallus turcicus) Leading to Increasing Female Chicks Proportion

Female birds of egg-laying hens are more valuable than males, because males bird have no commercial value. The opportunity to manipulate offspring sex ratio in poultry has been observed in female birds; hence, female birds have heterogametic ZW chromosomes. High progesterone concentration 2-5 h before ovulation is thought to affect the segregation of female sex chromosomes as the basis for determining the sex of offspring. This study aimed to investigate the effect of progesterone induction in Arabic hens, which was conducted 5 h before ovulation, along with the time of the first meiosis; therefore, segregation of sex chromosomes leading to an increase in female offspring. The study utilized on 60 Arabic hens divided into three treatment groups: Without injection (P0), progesterone injection 1 mg/hen, (P1), progesterone injection 2 mg/hen (P2). The results showed that the percentage of female offspring at P0 and P1 was 54.74 and 53.33%, respectively, while P2 hens resulted in 62.92% females, the highest female offspring rate than the other groups. Chi-square analysis of sex ratio showed that P0 and P1 hens had no significant effect (P>0.05) while P2 hens being significant (P<0.05) from the expected sex ratio (50:50). Progesterone concentration of hens produced female offspring in the P2 group was 7.15 ng/ml being highly significant (P<0.05) than that found in the hens produced male offspring (4.53 ng/ml). Progesterone 5 h preceding ovulation in the Arabic hens with a 2 mg/hen dose can increase the proportion of female offspring; therefore, an opportunity for sex ratio manipulation in Arabic chickens.

Effects of dietary cortisol administration on sex ratios in dwarf gouramis and rosy barbs

AbstractSexual differentiation in teleosts is based on diverse and complex combinations of genetic and environmental factors. Environmental conditions during early development can alter gonadal differentiation leading to sex reversal, where the individual's genotypic and phenotypic sexes are opposite. In many species, the mechanism of environmental sex reversal has been linked to the stress axis and fluctuations in cortisol. This mechanism could be utilized to manipulate sex ratios in ornamental aquaculture, where the males of sexually dichromic species are often more economically valuable due to their brighter coloration. Dietary cortisol administration was used as a proxy for environmental stress to evaluate the effects on gonadal differentiation in two ornamental species, rosy barb, Pethia conchonius and dwarf gourami, Trichogaster lalius. Three experiments were run with each species to assess the following: (a) postprandial cortisol levels following dietary administration, (b) the effect of dietary cortisol concentration on sex ratios, and (c) the effect of developmental timing of cortisol feed administration on sex ratios. Whole‐body cortisol enzyme‐linked immunosorbent assays confirmed that cortisol feed did cause increased cortisol levels. However, despite cortisol feed being administered during the putative gonadal differentiation period, no effect was observed in the final sex ratios for any of the treatments evaluated.

Reintroducing Cuvier’s gazelle. Better than expected from captive-bred founders

The use of captive-bred animals as founder stock for reintroduction is sometimes discouraged due to their low genetic diversity and even accumulation of deleterious alleles. In October 2016, 43 Cuvier’s gazelles (12 males and 31 females), managed under a European Endangered Species Programme (EEP) and housed in captivity, formed the base of a reintroduction project in Tunisia. The project approach used soft-release techniques. Upon arrival, animals were released in acclimatisation pens, so they could adjust to the new conditions gradually before true release. This study reports on the three-year period gazelles were in the acclimatisation pens before release into the wild. To assess the suitability of captive-bred Cuvier’s gazelles as founder stock for reintroduction, the demographic parameters of the reintroduced population were studied for three reproductive seasons in Tunisia (2017–2019). The demographic parameters of the reintroduced population were also compared to those from the source captive population as a control during the first breeding season. If the animals used as founders were unsuited, a decrease in demographic parameters could be expected over time in the reintroduced population, as well as lower demographic variables compared to the source population. Contrary to expectations, during the three-year study period, all demographic variables increased in the population reintroduced in Tunisia, except juvenile mortality, which decreased. Moreover, none of the demographic values of the gazelles in Tunisia were significantly different from the source population. We hypothesize that in the extremely bottlenecked captive Cuvier’s gazelle population used as founder stock, genetic diversity was still high enough to surmount the presumably deleterious effects of inbreeding. This is probably due to very high heritability (h2), a parameter providing information on the quantitative genetic variation associated with multi-locus quantitative traits previously found in this population. Although reintroduction programs have traditionally been undertaken purely as management exercises, ours was designed to meet a research objective as well. We wanted to find out the adaptive variation in sex ratio in offspring of female Cuvier’s gazelles using the size of the enclosure as a proxy reflecting their body condition. As found in captivity using consanguinity level as a proxy of body condition, in the reintroduced population, mothers in large enclosures produced more daughters while those in the small ones produced more sons, which supports adaptive manipulation of the birth sex ratio favoured by natural selection in reintroduced Cuvier’s gazelles.

No evidence of sex ratio manipulation by Galápagos mockingbirds in response to environment

Birds can adjust investment in reproduction by altering the sex ratios of their broods. When one sex is more costly to raise than the other, producing either more male or female offspring can lower the costs of reproduction. Biasing sex ratios can also be advantageous if the expected fitness of male versus female offspring differs. Here, we investigated whether Galápagos mockingbirds Mimus parvulus bias sex ratios in response to variation in rainfall. Female mockingbirds are smaller and thus females may reduce the costs of reproduction by biasing sex ratios in favor of female offspring. We quantified sex ratios in 131 mockingbird broods over four years with variable rainfall (2012, 2013, 2015 and 2016). We found no evidence that mockingbirds adjusted sex ratios in response to environmental conditions. Mockingbirds may not adjust sex ratios either because they lack the ability to do so, or because the costs of manipulation outweigh its benefits.

Effects of larval diets and temperature regimes on life history traits, energy reserves and temperature tolerance of male Aedes aegypti (Diptera: Culicidae): optimizing rearing techniques for the sterile insect programmes

BackgroundProducing high quality sterile males is vital in Aedes aegypti rear-and-release birth control strategies. Larval diets, rearing temperatures, and their interactions determine the accumulation rates of essential nutrients in larvae, but these factors have been understudied in relation to mass-rearing techniques for producing eminent males.MethodsWe compared the effects of two larval diets, a cereal-legume-based diet (Khan’s diet) and a standard larval diet developed in the FAO/IAEA Insect Pest Control Laboratory (IAEA 2 diet). Diets were tested at selected temperatures for both larval and male adult life history traits, adult extreme temperature tolerance, and mating capacity relative to energy reserves of reared male adult Ae. aegypti.ResultsKhan’s diet resulted in shorter immature development time at each test temperature (except for 25 °C) than an IAEA 2 diet. Larvae reared at 28 °C and 32 °C with Khan’s diet demonstrated low pupation rates (c.80%). We accounted for these phenomena as secondary sex ratio manipulation, because a higher proportion of male adults emerged at 28 °C and 32 °C than that for the IAEA 2 diet. In general, the pupal development time shortened as temperature increased, resulting in higher teneral energy reserves in male mosquitoes. High energy reserves allowed male mosquitoes reared with Khan’s diet to have higher adult longevity (5–6 days longer when sugar-fed and 2–3 days longer when water-fed) and tolerance of heat stress than those fed on the IAEA 2 diet. The IAEA 2 diet produced larger male mosquitoes than Khan’s diet did: mosquitoes fed on Khan’s diet were 1.03–1.05 times smaller than those fed on the IAEA 2 diet at 28 °C and 32 °C. No evidence indicated reduced mating capacity for small mosquitoes fed on Khan’s diet.ConclusionsLarvae reared at 28 °C and 32 °C with Khan’s diet were characterized by shorter immature development time compared with those fed on the IAEA 2 diet. Adult mosquitoes produced from that larval rearing condition exhibited a significant male bias, long lifespan, and better endurance against extreme temperatures relative to energy reserves. Thus, the larval diet at rearing temperature of 28 °C and 32 °C optimized rearing techniques for the sterile insect programmes. However, mating competitiveness and flight performance of adult males require further investigation.

Effect of male age structure on reproduction in white‐tailed deer

ABSTRACTSelective harvest regimes that create female‐biased sex ratios can potentially lead to delayed breeding, reduced breeding synchrony, reduced productivity, and a female‐biased sex ratio of offspring. These resulting changes in breeding behavior and population dynamics have potential to adversely affect population growth. In 2002, Pennsylvania implemented harvest regulation changes that reduced deer density (increased harvest of antlerless deer) and increased the number and age of antlered deer (implemented antler point restriction regulations) that resulted in a less female‐biased sex ratio. We monitored date of conception, productivity (embryos/female), and sex ratio of embryos during 1999–2006 to test if timing of breeding occurred earlier and with greater synchrony, if productivity of females increased, and if the sex ratio of offspring would shift towards more males. Deer density decreased 23% and the adult (≥1.5 yr old) sex ratio declined from 2.30 to 1.95 females/male. The ratio of ≥2.5‐year‐old to 1.5‐year‐old males shifted towards more older males (1:3.7 in 2002 to 1:1.59 in 2006) and the ≥2.5‐year‐old male population increased from 41,853 during 1999–2001 to 54,064 by 2006. We found no evidence of any change in the timing or variability of date of conception, productivity, or offspring sex ratio. We conclude that harvest regulation changes implemented in Pennsylvania, USA, were insufficient to affect timing of breeding or population dynamics and that efforts by managers to identify a desired sex ratio or manipulate sex ratios to achieve management goals on a statewide scale will be challenging. © 2019 The Wildlife Society.