Sperm Numbers as a Paternity Guard in a Wild Bird

Sperm competition is thought to impose strong selection on males to produce competitive ejaculates to outcompete rival males under competitive mating conditions. Our understanding of how different sperm traits influence fertilization success, however, remains limited, especially in wild populations. Recent literature highlights the importance of incorporating multiple ejaculate traits and pre-copulatory sexually selected traits in analyses aimed at understanding how selection acts on sperm traits. However, variation in a male’s ability to gain fertilization success may also depend upon a range of social and ecological factors that determine the opportunity for mating events both within and outside of the social pair-bond. Here, we test for an effect of sperm quantity and sperm size on male reproductive success in the red-back fairy-wren (Malurus melanocephalus) while simultaneously accounting for pre-copulatory sexual selection and potential socio-ecological correlates of male mating success. We found that sperm number (i.e., cloacal protuberance volume), but not sperm morphology, was associated with reproductive success in male red-backed fairy-wrens. Most notably, males with large numbers of sperm available for copulation achieved greater within-pair paternity success. Our results suggest that males use large sperm numbers as a defensive strategy to guard within-pair paternity success in a system where there is a high risk of sperm competition and female control of copulation. Finally, our work highlights the importance of accounting for socio-ecological factors that may influence male mating opportunities when examining the role of sperm traits in determining male reproductive success.

Selection on Sperm Count, but Not on Sperm Morphology or Velocity, in a Wild Population of Anolis Lizards

Sperm competition is a widespread phenomenon that shapes male reproductive success. Ejaculates present many potential targets for postcopulatory selection (e.g., sperm morphology, count, and velocity), which are often highly correlated and potentially subject to complex multivariate selection. Although multivariate selection on ejaculate traits has been observed in laboratory experiments, it is unclear whether selection is similarly complex in wild populations, where individuals mate frequently over longer periods of time. We measured univariate and multivariate selection on sperm morphology, sperm count, and sperm velocity in a wild population of brown anole lizards (Anolis sagrei). We conducted a mark-recapture study with genetic parentage assignment to estimate individual reproductive success. We found significant negative directional selection and negative quadratic selection on sperm count, but we did not detect directional or quadratic selection on any other sperm traits, nor did we detect correlational selection on any trait combinations. Our results may reflect pressure on males to produce many small ejaculates and mate frequently over a six-month reproductive season. This study is the first to measure multivariate selection on sperm traits in a wild population and provides an interesting contrast to experimental studies of external fertilizers, which have found complex multivariate selection on sperm phenotypes.

Male sperm storage impairs sperm quality in the zebrafish

Variation in sperm traits is widely documented both at inter- and intraspecific level. However, sperm traits vary also between ejaculates of the same male, due for example, to fluctuations in female availability. Variability in the opportunities to mate can indeed have important consequences for sperm traits, as it determines how often sperm are used, and thus the rate at which they are produced and how long they are stored before the mating. While being stored within males’ bodies, sperm are subjected to ageing due to oxidative stress. Sperm storage may significantly impair sperm quality, but evidence linking male sperm storage and variation in sperm traits is still scarce. Here, we tested the effect of the duration of sperm storage on within-male variation in sperm traits in the zebrafish, Danio rerio. We found that without mating opportunities, sperm number increased as storage duration increased, indicating that sperm continue to be produced and accumulate over time within males without being discharged in another way. Long sperm storage (12 days) was associated with an overall impairment in sperm quality, namely sperm motility, sperm longevity, and sperm DNA fragmentation, indicating that sperm aged, and their quality declined during storage. Our results confirm that male sperm storage may generate substantial variation in sperm phenotype, a source of variation which is usually neglected but that should be accounted for in experimental protocols aiming to assay sperm traits or maximise fertilization success.

Lipid peroxidation in bull semen influences sperm traits and oxidative potential of Percoll®-selected sperm

Summary Although bovine embryo in vitro production (IVP) is a common assisted reproductive technology, critical points warrant further study, including sperm traits and oxidative status of sperm for in vitro fertilization (IVF). Our aim was to evaluate whether the lipid peroxidation index of commercial bull semen is influenced by sperm traits and oxidative status of sperm populations selected using Percoll® gradient. Semen straws from 48 batches from 14 Nelore bulls were thawed individually, analyzed for motility and subjected to Percoll selection. After Percoll, the lipid peroxidation index of the extender was evaluated, whereas selected sperm were analyzed for motility, acrosome and membrane integrity, mitochondrial membrane potential, chromatin resistance and oxidative potential under IVF conditions. Batches were divided retrospectively in four groups according to lipid peroxidation index. Sperm from Group 4 with the lowest index of lipid peroxidation had, after Percoll selection, greater plasma membrane integrity (81.3%; P = 0.004), higher mitochondrial potential (81.1%; P = 0.009) and lower oxidative potential (135.3 ng thiobarbituric acid reactive substances (TBARS)/ml; P = 0.026) compared with Group 1 with highest lipid peroxidation index (74.3%, 73% and 213.1 ng TBARS/ml, respectively). Furthermore, we observed negative correlations for the lipid peroxidation index with motility, membrane integrity and mitochondrial potential, and positive correlations with oxidative potential. In conclusion, oxidative stress in semen straws, as determined using lipid peroxidation in the extender, is associated with sperm traits and their oxidative potential under IVF conditions. These results provided further insights regarding the importance of preventing oxidative stress during semen handling and cryopreservation, as this could affect sperm selected for IVF. Finally, Percoll selection did not completely remove sperm with oxidative markers.

Measuring Pre- and Post-Copulatory Sexual Selection and Their Interaction in Socially Monogamous Species with Extra-Pair Paternity

When females copulate with multiple males, pre- and post-copulatory sexual selection may interact synergistically or in opposition. Studying this interaction in wild populations is complex and potentially biased, because copulation and fertilization success are often inferred from offspring parentage rather than being directly measured. Here, I simulated 15 species of socially monogamous birds with varying levels of extra-pair paternity, where I could independently cause a male secondary sexual trait to improve copulation success, and a sperm trait to improve fertilization success. By varying the degree of correlation between the male and sperm traits, I show that several common statistical approaches, including univariate selection gradients and paired t-tests comparing extra-pair males to the within-pair males they cuckolded, can give highly biased results for sperm traits. These tests should therefore be avoided for sperm traits in socially monogamous species with extra-pair paternity, unless the sperm trait is known to be uncorrelated with male trait(s) impacting copulation success. In contrast, multivariate selection analysis and a regression of the proportion of extra-pair brood(s) sired on the sperm trait of the extra-pair male (including only broods where the male sired ≥1 extra-pair offspring) were unbiased, and appear likely to be unbiased under a broad range of conditions for this mating system. In addition, I investigated whether the occurrence of pre-copulatory selection impacted the strength of post-copulatory selection, and vice versa. I found no evidence of an interaction under the conditions simulated, where the male trait impacted only copulation success and the sperm trait impacted only fertilization success. Instead, direct selection on each trait was independent of whether the other trait was under selection. Although pre- and post-copulatory selection strength was independent, selection on the two traits was positively correlated across species because selection on both traits increased with the frequency of extra-pair copulations in these socially monogamous species.

Spermatogenic Activity and Sperm Traits in Post-Pubertal and Adult Tomcats (Felis catus): Implication of Intra-Male Variation in Sperm Size

Tomcats are considered to be adults at 1 year of age, although many reach sexual maturity at an earlier age. Nevertheless, we still know little about whether the spermatogenic activity and sperm quality of mature under one-year-old tomcats differ from those of tomcats that are over one-year-old. This study aims to evaluate the spermatogenic activity, sperm traits, and their relationships in mature tomcats at two different ages. Sixteen tomcats showing complete spermatogenesis and spermatozoa in their epididymal caudae were used and classified according to their age as post-pubertal (<1 year old) or adult (˃1 year old). Our results show that adult cats have higher epididymal sperm concentration and lower coefficient of variation in sperm head width and ellipticity than post-pubertal cats. However, they do not differ in their testicular and epididymal mass, spermatogenesis, and sperm traits such as motility, mitochondrial activity, morphology, morphometry, as well as plasma membrane, acrosome, and DNA integrity. Reduced intra-male variation of sperm head ellipticity is associated with higher testis mass, epididymis mass, and sperm concentration. Interestingly, low intra-male variation in sperm head size is associated with increased Sertoli cell function and reduced post-meiotic germ cell loss. These findings increase our knowledge about feline reproductive physiology and provide new insights into the functional significance of low intra-male variation in sperm size and shape in tomcats.

Effects of NGF Addition on Llama (Lama glama) Sperm Traits After Cooling

To provide new insights into the mechanisms through which seminal plasma proteins can protect sperm from damage caused during refrigeration, we evaluate the possibility that β-NGF can contribute to the improvement of sperm quality after cooling. First, β-NGF was detected in refrigerated sperm and compared with unrefrigerated sperm by western blotting of the proteins adsorbed by sperm, showing that native β-NGF is still present even 24 h after cooling only as an active form. Then, the effect of exogenous β-NGF on sperm quality after cooling was evaluated. A total of 12 ejaculates from male llamas (three ejaculates per male), were obtained by electro-ejaculation, diluted 4:1 with buffer Hepes-balanced salt solution and centrifuged at 800 × g for 8 min to remove the seminal plasma. Sperm were suspended in Tris-citrate-fructose-egg yolk diluent for a final concentration of 30 ×106/ml and cooled at 5°C for 24 h. After refrigeration, the extended sperm were equilibrated for 5 min at 37°C and divided into the following subgroups: sperm samples without treatment (control) and sperm samples supplemented with exogenous human β-NGF (10, 100, and 500 ng/ml). At 5, 30, and 60 min of incubation sperm were evaluated for sperm viability (using eosin/nigrosin stain), sperm motility and vigor (observed under light microscopy), and mitochondrial activity (using the JC-1 fluorescent marker). Vigor data were analyzed with the nonparametric Kruskal-Wallis test. The rest of the variables were analyzed with a mixed models approach. Mean comparisons were performed using Fisher's LSD test with a confidence level of 95%. A principal components analysis was performed to analyze the relationships between variables. Treatment of 24 h cooled sperm with 10 or 100 ng/ml of human β-NGF increased the percentage of total motility and vigor (p &lt; 0.05). Besides, an incubation time of 60 min would be adequate to improve sperm quality, since all variables are positively related. The significant improvement observed in the motility and vigor of post-refrigerated sperm suggests that supplementation with exogenous β-NGF may be profitable for the improvement of cooled llama sperm.

Effect of ESR, FSHB and PRLR Genes on Sperm Traits of Landrace and Yorkshire Boars in the Tropical Environmental Conditions of Vietnam

Background: Fertility traits in pigs made a restricted progress through traditional selection. Applying marker assisted selection could improve these traits. The aim of the study was to investigate the effects of candidate genes Estrogen Receptor (ESR), Follicle Stimulating Hormone Beta (FSHB) and Prolactin Receptor (PRLR) on sperm quality traits of Landrace and Yorkshire boars under tropical conditions in Northern Vietnam.Methods: A total of 6306 ejaculates from 140 boars (92 Landrace and 48 Yorkshire) were collected to estimate sperm ejaculate volume (VOL), spermatozoon motility (MO), sperm concentration (CO) and total number of spermatozoon in ejaculate (NT). Ear tissue samples were collected for genotype identification of SNP present in ESR, FSHB and PRLR genes using PCR-RFLP technique. A mixed model was used to test the effect of different genetic and non-genetic factors on the sperm quality traits.Result: The genotype frequencies of ESR, FSHB and PRLR were in Hardy-Weinberg equilibrium for each breed. Age of boars and month of the year had significant effect on all the sperm quality traits (P less than 0.01) while effect of breed was found to be non-significant on all the sperm quality trait. FSHB gene significantly (P less than 0.05) affected VOL, MO and CO. Boars with BB genotype showed positive effect on VOL but negative effect on MO and CO in comparison with AA genotype boars. ESR gene showed only effect on VOL while PRLR affected only MO. Boars with AA and AB genotypes of ESR gene had a significantly higher VOL than those with BB genotype (P less than 0.05). For PRLR gen, AB genotype was associated with higher MO than AA genotype (P less than 0.05). These results suggest a possibility of using the existing polymorphisms in ESR, FSHB and PRLR genes to improve some sperm traits of Landrace and Yorkshire boars.