Ranking parameters driving siring success during sperm competition in the North African houbara bustard.

Sperm competition is a powerful force driving the evolution of ejaculate and sperm traits. However, the outcome of sperm competition depends on many traits that extend beyond ejaculate quality. Here, we study male North African houbara bustards (Chlamydotis undulata undulata) competing for egg fertilization, after artificial insemination, with the aim to rank the importance of 14 parameters as drivers of siring success. Using a machine learning approach, we show that traits independent of male quality (i.e., insemination order, delay between insemination and egg laying) are the most important predictors of siring success. Traits describing intrinsic male quality (i.e., number of sperm in the ejaculate, mass motility index) are also positively associated with siring success, but their contribution to explaining the outcome of sperm competition is much lower than for insemination order. Overall, this analysis shows that males mating at the last position in the mating sequence have the best chance to win the competition for egg fertilization. This raises the question of the importance of female behavior as determinant of mating order.

Genetic diversity and relatedness amongst captive saker falcons (Falcocherrug) in the Green Balkans' Wildlife Rehabilitation and Breeding Centre in Bulgaria.

The globally endangered saker falcon (Falcocherrug) is currently being re-introduced in Bulgaria, where the falcons are bred in captivity and released through the hacking method. We relied on the birds' pedigree when forming the breeding pairs from 2011. In 2021-2022, we had the opportunity to evaluate our captive population via DNA tests. We performed the first genetic assessment of the sakers in the WRBC through a genome evaluation of the most important founders (n = 12) and, in 2022, we executed a microsatellite analysis on 30 saker falcons from the programme. We compared the results with the known pedigree and history of the saker falcons. The genetic tests helped to assign relatedness to some birds with missing or incomplete pedigrees, indicating the test can complement that information and lead to better management of the captive group. One pair was separated as a precaution as it was indicated by one the tests that the two birds are more closely related than expected. The research could be beneficial to other raptor captive breeding programmes dealing with a similar group composition.

Assisted Reproduction Techniques to Improve Reproduction in a Non-Model Species: The Case of the Arabian Bustard (Ardeotis arabs) Conservation Breeding Program.

Artificial reproductive technologies are highly valuable for ex situ conservation. While Arabian bustard populations are declining and extinct in some parts of the range, the International Fund for Houbara Conservation in the United Arab Emirates implemented a conservation breeding program. Since 2012, a total of 1253 eggs were laid through natural reproduction, 1090 were incubated and 379 of these were fertile (fertility rate of 34.8%), leading to the production of 251 chicks. To improve fertility and acquire crucial knowledge for other endangered large birds, artificial reproduction was implemented in 2018 using fresh, refrigerated, and frozen sperm. A total of 720 ejaculates were collected from 12 birds. We analysed these samples for concentration, volume, motility score (0 to 5), viability (eosin/nigrosine), length, and morphology. The first age at collection was 35.7 ± 18.8 months, mean volume was 89.2 ± 65.3 µL, mean concentration was 928 ± 731 sptz/mL and mean motility score was 2.61 ± 0.95. Morphology analyses revealed a bimodal distribution of sperm length. Five hundred and thirty-five ejaculates were cryopreserved and the initial motility score was 3.4 ± 0.7 and 2.0 ± 0.6 after thawing, while the percentage of normal and intact membrane sperm cells decreased from 88.8 ± 7.5% to 52.9 ± 1%. Sixty-five artificial inseminations were performed, leading to a global fertility rate of 84.3%-more precisely, 85.2% and 83.3%, respectively, for fresh and cryopreserved semen. All methods successfully produced fertile eggs, indicating that artificial insemination is an efficient tool for the conservation and genetic management of the species.

Enforced monoandry over generations induces a reduction of female investment into reproduction in a promiscuous bird.

While uncovering the costs and benefits of polyandry has attracted considerable attention, assessing the net effect of sexual selection on population fitness requires the experimental manipulation of female mating over generations, which is usually only achievable in laboratory populations of arthropods. However, knowing if sexual selection improves or impairs the expression of life-history traits is key for the management of captive populations of endangered species, which are mostly long-lived birds and mammals. It might therefore be questionable to extrapolate the results gathered on laboratory populations of insects to infer the net effect of sexual selection on populations of endangered species. Here, we used a longitudinal dataset that has been collected on a long-lived bird, the houbara bustard, kept in a conservation breeding program, to investigate the effect of enforced monoandry on female investment into reproduction. In captivity, female houbara bustards are artificially inseminated with sperm collected from a single male (enforced monoandry), or sequentially inseminated with semen of different males (polyandry), allowing postcopulatory sexual selection to operate. We identified female lines that were produced either by monoandrous or polyandrous inseminations over three generations, and we compared reproductive investment of females from the two mating system groups. We found that females in the polyandrous lines had higher investment into reproduction as they laid more eggs per season and produced heavier hatchlings. Higher reproductive investment into reproduction in the polyandrous lines did not result from inherited differences from females initially included in the two mating system groups. These results show that removal of sexual selection can alter reproductive investment after only few generations, potentially hindering population fitness and the success of conservation breeding programs.

Reproductive performance in houbara bustard is affected by the combined effects of age, inbreeding and number of generations in captivity.

Although captive breeding programs are valuable for conservation, they have been shown to be associated with genetic changes, such as adaptation to captivity or inbreeding. In addition, reproductive performance is strongly age-dependent in most animal species. These mechanisms that potentially impact reproduction have often been studied separately, while their interactions have rarely been addressed. In this study, using a large dataset of nine male and female reproductive parameters measured for 12,295 captive houbara bustards (Chlamydotis undulata undulata) over 24 years, we investigated the relative and interactive effects of age, inbreeding and number of generations in captivity on reproduction. We clearly identified (1) senescence patterns in all parameters studied; (2) negative effects of inbreeding on sperm characteristics, display behavior, egg weight, egg volume and hatching probability; and (3) changes in phenotypic values for seven parameters according to number of generations in captivity. However, the effect sizes associated with age were substantially greater than those associated with inbreeding and number of generations in captivity. Beyond the independent effects of these three factors on reproductive parameters, the results highlighted their interactive effects and thus the importance of integrating them in the design of genetic management plans for conservation breeding programs.

Paternal age negatively affects sperm production of the progeny.

Parental age has profound consequences for offspring's phenotype. However, whether patrilineal age affects offspring sperm production remains unknown, despite the importance of sperm production for male reproductive success in species facing post-copulatory sexual selection. Using a longitudinal dataset on ejaculate attributes of the houbara bustard, we showed that offspring sired by old fathers had different age-dependent trajectories of sperm production compared to offspring sired by young fathers. Specifically, they produced less sperm (-48%) in their first year of life, and 14% less during their lifetime. Paternal age had the strongest effect, with weak evidence for grandpaternal or great grandpaternal age effects. These results show that paternal age can affect offspring reproductive success by reducing sperm production, establishing an intergenerational link between ageing and sexual selection.

Genetic assessment of a conservation breeding program of the houbara bustard (Chlamydotis undulata undulata) in Morocco, based on pedigree and molecular analyses.

Protection and restoration of species in the wild may require conservation breeding programs under genetic management to minimize deleterious effects of genetic changes that occur in captivity, while preserving populations' genetic diversity and evolutionary resilience. Here, through interannual pedigree analyses, we first assessed the efficiency of a 21-year genetic management, including minimization of mean kinship, inbreeding avoidance, and regular addition of founders, of a conservation breeding program targeting on Houbara bustard (Chlamydotis undulata undulata) in Morocco. Secondly, we compared pedigree analyses, the classical way of assessing and managing genetic diversity in captivity, to molecular analyses based on seven microsatellites. Pedigree-based results indicated an efficient maintenance of the genetic diversity (99% of the initial genetic diversity retained) while molecular-based results indicated an increase in allelic richness and an increase in unbiased expected heterozygosity across time. The pedigree-based average inbreeding coefficient F remained low (between 0.0004 and 0.003 in 2017) while the proportion of highly inbred individuals (F > .1) decreased over time and reached 0.2% in 2017. Furthermore, pedigree-based F and molecular-based individual multilocus heterozygosity were weakly negatively correlated, (Pearson's r = -.061 when considering all genotyped individuals), suggesting that they cannot be considered as alternatives, but rather as complementary sources of information. These findings suggest that a strict genetic monitoring and management, based on both pedigree and molecular tools can help mitigate genetic changes and allow to preserve genetic diversity and evolutionary resilience in conservation breeding programs.

Post-copulatory sexual selection allows females to alleviate the fitness costs incurred when mating with senescing males.

Male senescence has detrimental effects on reproductive success and offspring fitness. When females mate with multiple males during the same reproductive bout, post-copulatory sexual selection that operates either through sperm competition or cryptic female choice might allow females to skew fertilization success towards young males and as such limit the fitness costs incurred when eggs are fertilized by senescing males. Here, we experimentally tested this hypothesis. We artificially inseminated female North African houbara bustards with sperm from dyads of males of different (young and old) or similar ages (either young or old). Then, we assessed whether siring success was biased towards young males and we measured several life-history traits of the progeny to evaluate the fitness costs due to advanced paternal age. In agreement with the prediction, we found that siring success was biased towards young males, and offspring sired by old males had impaired hatching success, growth and post-release survival (in females). Overall, our results support the hypothesis that post-copulatory sexual selection might represent an effective mechanism allowing females to avoid the fitness costs of fertilization by senescing partners.

Sperm competition accentuates selection on ejaculate attributes.

Ejaculate attributes are important factors driving the probability of fertilizing eggs. When females mate with several males, competition between sperm to fertilize eggs should accentuate selection on ejaculate attributes. We tested this hypothesis in the North African houbara bustard ( Chlamydotis undulata undulata) by comparing the strength of selection acting on two ejaculate attributes when sperm from single males or sperm from different males were used for insemination. In agreement with the prediction, we found that selection on ejaculate attributes was stronger when sperm of different males competed for egg fertilization. These findings provide the first direct comparison of the strength of selection acting on ejaculate attributes under competitive and non-competitive fertilizations, confirming that sperm competition is a major selective force driving the evolution of ejaculate characteristics.

No evidence for prezygotic postcopulatory avoidance of kin despite high inbreeding depression.

Offspring resulting from mating among close relatives can suffer from impaired fitness through the expression of recessive alleles with deleterious effects. Postcopulatory sperm selection (a prezygotic mechanism of cryptic female choice) has been suggested to be an effective way to avoid inbreeding. To investigate whether postcopulatory female choice allows the avoidance of fertilization by close kin, we performed artificial inseminations in a promiscuous bird, the houbara bustard (Chlamydotis undulata undulata). Females were inseminated with a mix of sperm from triads of males, each constituted of a male genetically unrelated to the female, a first cousin and a half-sibling. When counting the number of eggs sired by unrelated males, cousins or half-siblings, we found a significant deviation from the expected random distribution, with more eggs sired by unrelated males. However, offspring sired by cousins, and especially by half-siblings suffered from high prehatching mortality, suggesting that the observed paternity skew towards unrelated males might reflect differential offspring mortality rather than sperm selection. In agreement with this hypothesis, within-triad siring success was similar for the three parental relatedness categories, but the relationship between siring and hatching success differed across categories. In clutches with high hatching success, unrelated males had the highest success while in clutches with high failure rate, half-siblings had the highest success. Offspring sired by half-siblings also suffered from reduced growth rate during the first three months and higher posthatching mortality. Hence, despite substantial fitness costs associated with fertilization by close relatives, females do not seem to select sperm of unrelated males.

Does recognized genetic management in supportive breeding prevent genetic changes in life-history traits?

Supportive breeding is one of the last resort conservation strategies to avoid species extinction. Management of captive populations is challenging because several harmful genetic processes need to be avoided. Several recommendations have been proposed to limit these deleterious effects, but empirical assessments of these strategies remain scarce. We investigated the outcome of a genetic management in a supportive breeding for the Houbara Bustard. At the phenotypic level, we found an increase over generations in the mean values of gamete production, body mass and courtship display rate. Using an animal model, we found that phenotypic changes reflected genetic changes as evidenced by an increase in breeding values for all traits. These changes resulted from selection acting on gamete production and to a lesser extent on courtship display. Selection decreased over years for female gametes, emphasizing the effort of managers to increase the contribution of poor breeders to offspring recruited in the captive breeding. Our results shed light on very fast genetic changes in an exemplary captive programme that follows worldwide used recommendations and emphasizes the need of more empirical evidence of the effects of genetic guidelines on the prevention of genetic changes in supportive breeding.