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Protamines are proteins responsible for condensing sperm chromatin. There are two protamines whose ratio remains constant in each species and which is related to fertility. To quantify their expression, it is necessary to have a good protocol of sample collection (i.e., RNA stabilizing buffers and temperature conditions). The aim of this work was to compare gene expression of protamines, with analysis of RNA quality and ratios, in testis samples from wild-derived mice, Mus musculus, preserved in different buffers (RNAlater® or Nucleic Acid Preservation-NAP-buffer) and different temperatures (room temperature -RT-, 4°C, -20°C, -80°C or liquid nitrogen) for different times (one week, one month, 3 months and one year). The relative abundance of protamine expression was assessed by qPCR using 18S rRNA as housekeeping. The results showed that the preservation of testes in RNAlater® or NAP buffer at -80°C afforded equivalent good preservation as in somatic tissues. Testis samples stored at RT in both buffers for 1 week resulted in a similar RNA quality and protamine expression over time. Moreover, samples in RNAlater® stored at RT, 4°C, -20°C and -80°C, were analyzed after 24 h, 7 days, 30 days, 90 days or 365 days; samples stored at RT resulted in a loss of RNA quality but protamine ratio was maintained up to 90 days. Samples stored at 4°C and -20°C showed similar values of RNA integrity and protamine expression than those stored at -80°C. Finally, we stored testis samples at -80°C or -196°C, after initial snap-freezing in liquid nitrogen. Both methods afforded very good preservation of RNA integrity and protamine expression. These results open new possibilities for the collection, transport and storage of testes samples under field conditions., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright: © 2024 Latorre et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ovum Pick Up (OPU) is a minimally invasive technique widely used in cattle and mares for oocyte retrieval, involving ultrasound-guided puncture of ovarian follicles. It has been demonstrated that this technique is safe for its repeated use in the same female without affecting her reproductive health, allowing for the retrieval of oocytes in individuals regardless of their reproductive status. The oocytes obtained through OPU can subsequently be used for in vitro embryo production (IVP) using assisted reproductive techniques (ARTs) or be cryopreserved in biobanks for their future use. Traditionally, the minimally invasive technique of choice performed in vivo in domestic and wild felines was LOPU (laparoscopic-guided ovum pick up). The present study was designed to explore if ultrasound-guided OPU in the domestic cat is safe and effective. In an initial series of ex vivo experiments (n = 92 ovaries, n = 434 oocytes), the effect of different aspiration pressures for oocyte collection was explored. These experiments identified 43 mmHg as the optimal aspiration pressure, resulting in the highest recovery rate and a favorable maturation and blastocyst rate. Subsequently, 16 grade I and II oocytes were retrieved by OPU and 101 oocytes were retrieved following ovariectomy and slicing. Sixteen oocytes obtained with each technique were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). A total of 14 presumptive zygotes were selected for in vitro culture (IVC) from each group (OPU and slicing), obtaining a cleavage rate of 57.1 % and 64.2 %, a morula rate of 28.5 % in both groups, and a blastocyst rate of 7.14 % and 14.2 % respectively. The hormonal stimulation protocol was well-tolerated, with no adverse effects observed. Moreover, no complications arose during the ovariectomy performed post-OPU. The use of this technique in domestic cats represents a significant step forward in terms of safety, replicability, and invasiveness, serving as a valuable model for its application in wild felids species. Additional research involving a greater number of animals is required to validate these encouraging findings., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The administration of probiotics for the treatment of different diseases has gained interest in recent years. However, few studies have evaluated their effects on reproductive traits. The objective of this study was to examine the effect of two mixtures of probiotics, a commercial probiotic (Vivomixx®) and a mix of Lacticaseibacillus rhamnosus GG and Faecalibacterium duncaniae A2-165, on sperm quality in a mouse model. Adult male mice (8 months old) were used for two experimental and one control groups (n = 5 each). The probiotics or physiological serum (control) was administered orally, twice a week, during 5 weeks. Sperm were collected from the cauda epididymis, and their total number, motility, kinematics, morphology, and acrosome integrity were assessed in recently collected samples and after a 60-min in vitro incubation. Results showed a higher percentage of normal sperm in both experimental groups, with fewer head abnormalities than in the control. Differences were found among groups in the morphometry of sperm heads, being more elongated in mice treated with probiotics. Sperm from probiotic-treated mice showed similar total motility when compared to the controls, although the proportion of progressively moving sperm and their vigor of motility were lower. Sperm swimming descriptors were measured with a CASA system. Velocity parameters were similar among groups whereas linearity was higher in mice treated with the commercial probiotic. These results suggest that the administration of probiotics may increase the proportion of sperm with normal morphology and lead to modifications in sperm head shape that may enhance sperm swimming. Studies using a longer administration period would be useful in further characterizing the effect of these probiotic mixtures on sperm quality and fertilization capacity., (© 2024. The Author(s).)

This study investigated the optimization of assisted reproductive techniques for wild felid conservation, focusing on in vitro procedures using the domestic cat as a model species. The research evaluated the impact of three different in vitro culture media on blastocyst formation. Oocytes and spermatozoa were collected and processed, followed by in vitro fertilization and culture. Results returned a similar blastocyst rate (ANOVA, p > .05), over 16% across all groups. While demonstrating the potential of these techniques, further investigations are warranted to evaluate embryo quality to refine optimal protocols and their applicability in felid conservation efforts., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Bioenergetics plays a crucial role in sperm functions, including motility, capacitation-related protein modifications, oocyte recognition and interaction, all of which are essential for fertilization. Sperm metabolism is recognized as flexible, responding to environmental cues and energetic demands during ejaculation, the journey along the female tract, and until fertilization. Recent studies suggest that sperm metabolic functions are relevant beyond fertilization and may influence zygote and embryo development, impacting paternal-derived effects on offspring development and health. In recent years, sperm metabolic functions and homeostasis have gained increasing interest in male reproduction research. Given the crucial implications of sperm metabolism on fertility-related processes, this field is of interest not only in human male fertility but also in livestock research, semen conservation, and assisted reproductive techniques. Newly developed assessment tools are allowing a better understanding of sperm metabolism under different conditions and identifying species-specific peculiarities. This review aims to discuss the current knowledge of mammalian sperm metabolism, focusing on species-specific features, changes during the sperm journey, and potential contributions to translational research and reproductive biotechnologies. Furthermore, we propose future perspectives on sperm bioenergetics research., (© 2024 The Author(s). Molecular Reproduction and Development published by Wiley Periodicals LLC.)

This investigation aimed to analyze the effect that thawing time and temperature in combination with a termo-resistance test had on straws from dairy bulls used for artificial insemination (AI) on semen motility and kinematic variables measured with CASA systems. Eight animals of Holstein and Jersey breeds were used, and nine frozen-thawed semen doses per animal were analyzed for each breed. Three temperatures (35, 37, and 40 °C) and three thawing times (35, 40, and 45 s) were evaluated using a factorial design. Motility and kinematic patterns were analyzed using CASA-mot (Computer-Assisted Semen Analysis of motility) technology at different post-thawing times (0.5, 1, and 2 h). Sperm motility in Jersey bulls was higher ( p < 0.05) than in Holstein ones (64.52 ± 1.45% and 53.10 ± 1.40%, respectively). The same effect was seen with progressive motility among the two breeds (Jersey: 45.29 ± 1.00%; Holstein: 36.30 ± 0.98%, p < 0.05). The Jersey breed presented higher values ( p < 0.05) of curvilinear velocity (VCL), rectilinear velocity (VSL), average velocity (VAP), linearity on forward progression (LIN), and wobble (WOB). The Holstein breed showed a lower mean value ( p < 0.05) of the beat-cross frequency (BCF) compared to the Jersey breed, thus suggesting an effect on VCL and VAP. During the post-thaw period, a gradual increase in VCL was observed at 2 h. VSL and VAP showed a decrease ( p < 0.05) as the post-thaw period was prolonged. The study showed differences in sperm quality between Holstein and Jersey breeds, influenced by cryopreservation, thawing, and post-thawing incubation. Thawing at 37 °C for 30 s was considered optimal in relation to sperm motility. In addition, a decrease in sperm quality was observed as post-thawing time increased.

Background: Recently, the metabolic pathways involved in energy production and the role of aquaglyceroporins in capacitation-associated events have been studied in humans and mice. However, little is known about these in ram spermatozoa., Objective: The present study investigated bioenergetic and aquaglyceroporin 3 variations during in vitro capacitation of ram spermatozoa. In addition, differences in testosterone levels between males were examined to determine their influence on capacitation-like changes., Materials and Methods: Spermatozoa obtained from nine rams (ejaculates = 36) were incubated for 180 min in three different media (control, capacitating, and aquaglyceroporin-inhibitor media) at 38.5°C. At 0 and 180 min of incubation in each medium, sperm viability, kinetics, chlortetracycline patterns, adenosine triphosphate concentration, lactate excretion (final subproduct of glycolysis), and immunolocalization of aquaporin 3 were evaluated., Results: The increment of the capacitated spermatozoa-chlortetracycline pattern and the hyperactivated-like movement characterized by the highest curvilinear velocity and amplitude of lateral head displacement and the lowest linearity was only recorded after 180 min in the capacitating medium. At this time and conditions, adenosine triphosphate content and lactate excretion decreased, whereas the aquaglyceroporin 3 location in the midpiece and principal piece increased compared to 0 min. Such changes were not observed in the control medium over time. Incubation in the aquaglyceroporin-inhibitor medium for 180 min reduced drastically sperm motility and adenosine triphosphate content compared to the other media. Testosterone analysis revealed a significant individual variability, which was also present in all sperm parameters evaluated. Furthermore, testosterone was negatively correlated with adenosine triphosphate content but positively correlated with lactate excretion levels, sperm viability, motility, capacitated sperm-chlortetracycline pattern, and aquaglyceroporin 3 immunolabeling in the midpiece and principal piece., Conclusion: Despite individual differences, capacitation of ram spermatozoa increases adenosine triphosphate consumption, energy metabolism, and aquaglyceroporin 3 location in the midpiece and principal piece, which seems to be related to the acquisition of hyperactivated-like motility. Furthermore, testosterone levels may serve as a valuable tool to select those males with a greater sperm metabolism rate and fertilizing capacity., (© 2024 The Author(s). Andrology published by John Wiley & Sons Ltd on behalf of American Society of Andrology and European Academy of Andrology.)

Ethical considerations regarding our treatment of animals have gained strength, leading to legislation and a societal focus across various disciplines. This is a subject of study within curricula related to agri-food sciences. The aim was to determine the perceptions of agronomy university students concerning animal welfare in livestock production systems. A survey was conducted to encompass various aspects, from participants' sociodemographic attributes to their attitudes and behaviors regarding animal welfare and the consumption of animal products. Statistical analysis, performed using R software, delved into the associations between participants' characteristics and their perspectives on the ethical, bioethical, and legal dimensions of animal welfare. Associations between demographic factors and ethical viewpoints among students were identified. Gender differences emerged in animal treatment perceptions, while rural and urban environments impacted perspectives on various animals. Bioethical considerations revealed distinctive disparities based on gender and education in concerns regarding animal welfare, value perceptions, evaluations of animal behaviors, and opinions on animal research. It is crucial to distinguish between animal welfare and the ethical considerations arising from coexisting with sentient beings capable of experiencing suffering. Ethical theories provide a lens through which we perceive our obligations toward animals. The responsibility to ensure animal welfare is firmly rooted in recognizing that animals, like humans, experience pain and physical suffering. Consequently, actions causing unjustified suffering or mistreatment, particularly for entertainment purposes, are considered morally unacceptable.

The objective of the present study was to determine the effects that the reproductive season has on the motility, kinematics, morphology, and sperm morphometry of Brahman bulls evaluated with a commercial CASA system. The experiment was carried out at the Costa Rica Institute of Technology from March to August 2021. A total of eight Brahman bulls were used. A total of 28 ejaculates were collected in the pre-mating period (PMP), during it (DMP), and after it (AMP) using an electroejaculator. The sperm concentration was measured with the Accuread photometer. The motility was measured using a Spermtrack ® counting chamber. The analyses were performed with the CASA-Mot ISAS ® v1 system. The morphology was analyzed using a microscope with a negative phase contrast objective. Morphometry was evaluated with the CASA-Morph. The sperm concentration did not present differences between the PMP and AMP; however, it was significantly higher than DMP ( p > 0.05). Regarding the progressiveness variables, linearity on forward progression (LIN), straightness (STR), and wobble (WOB) were higher ( p < 0.05) DMP. A kinematic principal component analysis grouped all the variables into three factors and an effect on the reproductive period was found ( p < 0.05) in the parameters of the head and middle part of the sperm, such as width and perimeter, which were greater in the PMP. The length of the sperm head in the PMP and DMP did not show differences; however, both were larger ( p < 0.05) than AMP. The insertion distance of the middle piece of the sperm was significantly greater than DMP. Finally, the PMP contained cells with a larger insertion angle ( p < 0.05) than AMP. These findings are important to understand the implications of reproductive status on sperm quality and to consider them in andrological evaluations.

This study was designed to analyze changes in the spermatozoa of three species of Phodopus hamsters incubated under different conditions. Cauda epididymal sperm were incubated for 4 h in modified Tyrode's medium containing albumin, lactate, pyruvate, and Hepes (mTALP-H), in the same medium with the addition of bicarbonate (mTALP-BH), or with bicarbonate and 20 ng/mL of progesterone (mTALP-BH+P4). Media with bicarbonate are believed to promote capacitation in rodent species. Sperm motility, viability, capacitation patterns, and kinematics were assessed at different times. Capacitation in live cells was quantified after staining with Hoechst 33258 and chlortetracycline. Patterns believed to correspond to non-capacitated cells (F pattern), capacitated, acrosome-intact cells (B pattern), and acrosome-reacted cells (AR pattern) were recognized. Kinematics were examined via computer-assisted sperm analysis (CASA). The results showed a decrease in total motility in all three species in different media, with a sharp decrease in progressive motility in bicarbonate-containing media (without or with progesterone), suggesting hyperactivated motion. However, none of the other signs of hyperactivation described in rodents (i.e., decrease in STR or LIN, together with an increase in ALH) were observed. F pattern cells diminished with time in all media and were generally lower in P. roborovskii and higher in P. campbelli . B pattern cells increased in mTALP-BH media in all species. Progesterone did not enhance the percentage of B pattern cells. Finally, AR pattern cells increased in all species incubated in different media, showing the highest percentage in P. roborovskii and the lowest in P. campbelli . Comparisons between media revealed that there were higher percentages of F pattern cells and lower percentages of B pattern cells over time in medium without bicarbonate (mTALP-H) in comparison to media containing bicarbonate (mTALP-BH; mTALP-BH+P4). Overall, changes consistent with the acquisition of capacitation and development of hyperactivated motility were found; however, further studies are required to better characterize media necessary to support the pathways involved in these processes in Phodopus species.

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their intricate link to sperm function, embryo development, and overall fertility. Defects in chromatin compaction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, the chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus , M. spretus , and M. spicilegus . We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue, and chromomycin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a HEPES-buffered modified Tyrode's medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species employing diverse methodologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correlations with the percentages of sperm stained by aniline blue, methylene blue, and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species.

Enzootic bovine leukosis virus (BLV) and bovine herpesvirus 1 (BHV-1) are very important infectious agents for the livestock industry worldwide. The present study aimed to explore the association between natural exposure to BLV and BHV-1 with sperm quality analyzed by Computer-Assisted Semen Analysis (CASA) systems. Ten sexually mature Brahman bulls, with sanitary status BLV + /BHV-1 + (n = 2), BLV - /BHV-1 + (n = 6) and BLV - /BHV-1 - (n = 2) were evaluated twice, 30 days apart. Results showed that sanitary status of each bull was not associated with semen quality. It was found that the quality of the semen from the second collection was better due to the interruption of sexual rest. The evidence thus revealed that a bull infected with BLV generated good-quality contaminated semen and, therefore, that it is essential to detect contaminated seminal samples to prevent the spread of BLV. A multivariate analysis showed the presence of four sperm subpopulations in Brahman bulls that differ significantly in their kinematic patterns and with respect to sanitary status (P < 0.05), indicating that infection-free and seronegative bulls present the best kinematic parameters, which improved discrimination of sperm quality according to sanitary status. Overall, the analyses indicate that the seropositive-infected bulls with BLV and BHV-1 should be excluded from beef cattle farms., (© 2023. The Author(s).)

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 ( AK9 ) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

The identification of different morphometric patterns of spermatozoa serves as a basis for improving our understanding of the diversity in an ejaculate and to relate them to the potential fertility of males. In this study, we aimed to examine the semen subpopulation structure, following dilution in semen of extenders, using a mathematical approach a possible application to fertility analyses. Ten sexually mature Bos taurus bulls were randomly allotted to one of three groups: (1) Tris-citric acid-egg yolk extender (Tris-EY); (2) commercial egg yolk extender OptiXcell® and (3) commercial egg yolk extender Triladyl®. The results showed significant differences (p < .05) between extenders in terms of values for head size and head shape variables of individual sperm, indicating an influence of extender composition. Sperm head width was found to significantly differ (p < .05) according to the extender, decreasing in the following order: OptiXcell® (4.836 ± 0.017 μm), Triladyl® (4.695 ± 0.012 μm) and Tris-EY (4.638 ± 0.010 μm). Principal component analysis allowed us to identify two subpopulations in OptiXcell®, and three subpopulations were each found in Triladyl® and Tris-EY. Overall, we observed significant differences between sperm subpopulations within each extender (p < .05), with differences in sperm head size and shape between bovine species that can be related to functionality and fertility capabilities., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus., Competing Interests: The authors declare that MET received research support unrelated to this work from Boehringer-Ingelheim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Agudo-Rios, Rogers, King, Bhagat, Nguyen, Córdova-Fletes, Krapf, Strauss, Arévalo, Merges, Schorle, Roldan and Teves.)

Artificial insemination in the swine industry, as in other species, demands adequate semen handling and accurate evaluation for the preparation of seminal doses. Sperm concentration and motility estimates are part of the semen evaluation process and are considered important for maximizing the yield of doses for insemination. In this study, methods were examined for their accuracy in the estimation of boar sperm concentration and motility. Assessments of sperm concentration were carried out using iSperm ® , ISAS ® v1, Open CASA v2, and the Accuread ® photometer. Analyses of sperm motility were performed with iSperm ® , ISAS ® v1, and Open CASA v2 systems. In this study, boar semen samples were collected from 10 healthy males from two genetic lines. There were no relevant differences between sire lines when sperm concentration was assessed. A Bayesian analysis was applied to the four methods used to assess sperm concentration to examine whether there are relevant differences between them. Results suggested differences in the four methods, with a probability of relevance (PR) of 0.86-1.00. The iSperm ® method revealed higher concentration values within the highest posterior density region at 95% confidence interval (HPD 95% ) = 167.0, 224.2 M/mL, whereas Open CASA v2 showed the lowest values, with HPD 95% = 99.3, 155.9 M/mL. The iSperm ® demonstrated higher reliability in measuring sperm concentration compared to other methods or devices within the given range of confidence. ANOVAs revealed relevant differences in the three methods of motility estimation. Overall, differences in boar sperm concentration and motility estimates were found using various methods, but further studies are needed for better characterization of these differences.

Introduction: Human semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed., Methods: Seminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria., Results: The data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT

The acquisition of fertilizing ability by mammalian spermatozoa, known as "capacitation," includes processes that depend on particular metabolic pathways. This has led to the hypothesis that ATP demands might differ between capacitated and non-capacitated cells. Mouse sperm can produce ATP via OXPHOS and aerobic glycolysis, an advantageous characteristic considering that these cells have to function in the complex and variable environment of the female reproductive tract. Nonetheless, despite evidence showing that both metabolic pathways play a role in events associated with mouse sperm capacitation, there is contradictory evidence regarding changes promoted by capacitation in this species. In addition, the vast majority of studies regarding murine sperm metabolism use Mus musculus laboratory strains as model, thus neglecting the wide diversity of sperm traits of other species of Mus. Focus on closely related species with distinct evolutionary histories, which may be the result of different selective pressures, could shed light on diversity of metabolic processes. Here, we analyzed variations in sperm bioenergetics associated with capacitation in spermatozoa of the steppe mouse, Mus spicilegus, a species with high sperm performance. Furthermore, we compared sperm metabolic traits of this species with similar traits previously characterized in M. musculus. We found that the metabolism of M. spicilegus sperm responded to capacitation in a manner similar to that of M. musculus sperm. However, M. spicilegus sperm showed distinct metabolic features, including the ability to perform cross-pathway metabolic compensation in response to either respiratory or glycolytic inhibition, thus revealing a delicate fine-tuning of its metabolic capacities., (© 2022. The Author(s).)

In order to sustain motility and prepare for fertilization, sperm require energy. The characterization of sperm ATP production and usage in mouse species revealed substantial differences in metabolic pathways that can be differentially affected by capacitation. Moreover, spermatozoa encounter different environments with varying viscoelastic properties in the female reproductive tract. Here, we examine whether viscosity affects sperm ATP levels and kinematics during capacitation in vitro. Sperm from three mouse species ( Mus musculus , M. spretus , M. spicilegus ) were incubated under capacitating conditions in a modified Tyrode's medium containing bicarbonate, glucose, pyruvate, lactate, and bovine serum albumin (mT-BH) or in a bicarbonate-free medium as a non-capacitating control. Viscosity was increased with the inclusion of polyvinylpyrrolidone. ATP was measured with a bioluminescence kit, and kinematics were examined with a computer-aided sperm analysis system. In M. musculus sperm, ATP declined during capacitation, but no differences were found between non-capacitating and capacitating sperm. In contrast, in M. spretus and M. spicilegus , ATP levels decreased in capacitating sperm. Increasing viscosity in the medium did not modify the timing or proportion of cells undergoing capacitation but did result in additional time- and concentration-dependent decreases in ATP in M. spretus and M. spicilegus under capacitating conditions. Additionally, increased viscosity altered both velocity and trajectory descriptors. The limited impact of capacitation and higher viscosity on M. musculus sperm ATP and kinematics could be related to the low intensity of postcopulatory sexual selection in this species. Responses seen in the other two species could be linked to the ability of their sperm to perform better under enhanced selective pressures.

In mammals, sperm acquire fertilization ability after a series of physiological and biochemical changes, collectively known as capacitation, that occur inside the female reproductive tract. In addition to other requirements, sperm bioenergetic metabolism has been identified as a fundamental component in the acquisition of capacitation. Mammalian sperm produce ATP through two main metabolic processes, oxidative phosphorylation (OXPHOS) and aerobic glycolysis that are localized to two different flagellar compartments, the midpiece, and the principal piece, respectively. In mouse sperm, the occurrence of many events associated with capacitation relies on the activity of these two energy-producing pathways, leading to the hypothesis that some of these events may impose changes in sperm energetic demands. In the present study, we used extracellular flux analysis to evaluate changes in glycolytic and respiratory parameters of murine sperm that occur as a consequence of capacitation. Furthermore, we examined whether these variations affect sperm ATP sustainability. Our results show that capacitation promotes a shift in the usage ratio of the two main metabolic pathways, from oxidative to glycolytic. However, this metabolic rewiring does not seem to affect the rate at which the sperm consume ATP. We conclude that the probable function of the metabolic switch is to increase the ATP supply in the distal flagellar regions, thus sustaining the energetic demands that arise from capacitation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tourmente, Sansegundo, Rial and Roldan.)

Ejaculate quality can be regarded as multifactorial, with nutrition being a factor that could directly influence sperm parameters. The present study aimed to evaluate seminal quality associated with seasonal fat-soluble vitamin supplementation of boars. Seven sexually mature boars were randomly allotted to one of the three groups, and fed one of the three supplementary diets for 32 weeks: (1) control treatment (COD), without supplementation of fat-soluble vitamins, (2) treatment containing 100% fat-soluble vitamin supplementation administered intramuscularly, which was based on fat soluble vitamin supplementation (A, D3, E) (FVD1), and (3) treatment containing 50% of fat-soluble vitamin supplementation (FVD 1 2 ). Semen was collected at 7-day intervals. Semen samples were analyzed to assess several sperm parameters using the Computer-Assisted Semen Analysis (CASA) ISAS ® v1 system. Results showed that groups receiving FVD1 and FVD 1 2 supplementation had an increased semen volume. The percentages of motile and progressively motile sperm were increased by FVD1 treatment. A statistically significant interaction between treatment and season was found in the percentage of motility and progressive motility ( p < 0.05). Sperm concentrations showed significant differences ( p < 0.05) between treatments. Velocity variables (VSL, VCL, and VAP) were higher ( p < 0.05) in boars that received fat-soluble vitamin supplementation in comparison to controls receiving no supplementation. The FVD1 treatment presented spermatozoa with greater head size and more elongated heads ( p < 0.05). Overall, the utilization of dietary fat-soluble vitamin supplementation significantly improved the semen quality of boar ejaculates. This highlights the importance of fat-soluble vitamin supplementation in sexually active boars., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Calderón-Calderón, Sevilla, Roldan, Barquero and Valverde.)

Assessments of sperm performance are valuable tools for the analysis of sperm fertilizing potential and to understand determinants of male fertility. Hamster species constitute important animal models because they produce sperm cells in high quantities and of high quality. Sexual selection over evolutionary time in these species seems to have resulted in the largest mammalian spermatozoa, and high swimming and bioenergetic performances. Earlier studies showed that golden hamster sperm requires motility factors such as D-penicillamine, hypotaurine and epinephrine (PHE) to sustain survival over time, but it is unknown how they affect swimming kinetics or ATP levels and if other hamster species also require them. The objective of the present study was to examine the effect of PHE on spermatozoa of five hamster species ( Mesocricetus auratus , Cricetulus griseus , Phodopus campbelli , P. sungorus , P. roborovskii ). In sperm incubated for up to 4 h without or with PHE, we assessed motility, viability, acrosome integrity, sperm velocity and trajectory, and ATP content. The results showed differences in the effect of PHE among species. They had a significant positive effect on the maintenance of sperm quality in M. auratus and C. griseus , whereas there was no consistent effect on spermatozoa of the Phodopus species. Differences between species may be the result of varying underlying regulatory mechanisms of sperm performance and may be important to understand how they relate to successful fertilization.

Mammalian sperm differ widely in sperm morphology, and several explanations have been presented to account for this diversity. Less is known about variation in sperm physiology and cellular processes that can give sperm cells an advantage when competing to fertilize oocytes. Capacitation of spermatozoa, a process essential for mammalian fertilization, correlates with changes in motility that result in a characteristic swimming pattern known as hyperactivation. Previous studies revealed that sperm motility and velocity depend on the amount of ATP available and, therefore, changes in sperm movement occurring during capacitation and hyperactivation may involve changes in sperm bioenergetics. Here, we examine differences in ATP levels of sperm from three mouse species (genus Mus ), differing in sperm competition levels, incubated under non-capacitating and capacitating conditions, to analyse relationships between energetics, capacitation, and swimming patterns. We found that, in general terms, the amount of sperm ATP decreased more rapidly under capacitating conditions. This descent was related to the development of a hyperactivated pattern of movement in two species ( M. musculus and M. spicilegus ) but not in the other ( M. spretus ), suggesting that, in the latter, temporal dynamics and energetic demands of capacitation and hyperactivation may be decoupled or that the hyperactivation pattern differs. The decrease in ATP levels during capacitation was steeper in species with higher levels of sperm competition than in those with lower levels. Our results suggest that, during capacitation, sperm consume more ATP than under non-capacitating conditions. This higher ATP consumption may be linked to higher velocity and lateral head displacement, which are associated with hyperactivated motility.

The spermatozoon is a highly differentiated and polarized cell, with two main structures: the head, containing a haploid nucleus and the acrosomal exocytotic granule, and the flagellum, which generates energy and propels the cell; both structures are connected by the neck. The sperm's main aim is to participate in fertilization, thus activating development. Despite this common bauplan and function, there is an enormous diversity in structure and performance of sperm cells. For example, mammalian spermatozoa may exhibit several head patterns and overall sperm lengths ranging from ∼30 to 350 µm. Mechanisms of transport in the female tract, preparation for fertilization, and recognition of and interaction with the oocyte also show considerable variation. There has been much interest in understanding the origin of this diversity, both in evolutionary terms and in relation to mechanisms underlying sperm differentiation in the testis. Here, relationships between sperm bauplan and function are examined at two levels: first, by analyzing the selective forces that drive changes in sperm structure and physiology to understand the adaptive values of this variation and impact on male reproductive success and second, by examining cellular and molecular mechanisms of sperm formation in the testis that may explain how differentiation can give rise to such a wide array of sperm forms and functions.

Post-copulatory sexual selection is thought to influence the evolution of genes involved in reproduction. However, the detection of straightforward effects has been proven difficult due to the complexity and diversity of reproductive landscapes found in different taxa. Here, we compare the possible effect of relative testes mass as a sperm competition proxy on protamine genotype (protamine 1/protamine 2 ratio) and the link to sperm head phenotype in two rodent groups, mice, and voles. In mice, protamine expression ratios were found to increase from low values toward a 1:1 ratio in a positive association with testes mass, and relative sperm head area. In contrast, in voles, decreasing protamine expression ratios were found in species with larger testes but, surprisingly, they range from high values, again toward a 1:1 ratio, and showing a negative correlation with relative sperm head area. Altogether, we found differences in the way protamines seem to be selected and involved in adaptations of the sperm head in voles and mice. However, sexual selection driven by sperm competition seems to exhibit a common evolutionary pattern in both groups toward an equilibrium in the expression of the two protamines., (© 2021 The Authors. Evolution published by Wiley Periodicals LLC on behalf of The Society for the Study of Evolution.)

The aim was to determine the relationship between kinematic parameters of boar spermatozoa and fertility rates of sow, as well as to assess the effect of sperm clusters on the fertility capacity of the ejaculate. Semen samples were collected from 11 sexually mature boars. Samples were analyzed by an ISAS ® v1 CASA-Mot system for eight kinematic parameters. Ejaculate clusters were characterized using multivariate procedures, such as principal factors (PFs) analysis and clustering methods (the k-means model). Four different ejaculate clusters were identified from two kinematic PFs which involved linear trajectory and velocity. There were differences ( p < 0.05) between the sperm kinematic variables by sire line. There was no statistical difference ( p > 0.05) between dam lines and ejaculate clusters in fertility variables. The discriminant ability of the different kinematics of sperm variables to predict litter size fertility was analyzed using receiver operating characteristics (ROC) curve analysis. Curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF) showed significant, albeit limited, predictive capacity for litter size fertility variables (range: 0.55-0.58 area under curve, AUC). The kinematic analysis of the ejaculates in clusters did not have a predictive capacity for litter size variables.

The aim of the study was to compare the morphometric features of sperm head size and shape from the Pietrain line and the Duroc × Pietrain boar crossbred terminal lines, and to evaluate their relationship with reproductive success after artificial insemination of sows produced from crossbreeding the York, Landrace and Pietrain breeds. Semen samples were collected from 11 sexually mature boars. Only ejaculates with greater than 70% motility rate and <15% of abnormal sperm were used for artificial inseminations (AI) and included in the study. Samples were analyzed using an ISAS ® v1 computer-assisted sperm analysis system for eight morphometric parameters of head shape and size (CASA-Morph). Sub-populations of morphometric ejaculates were characterized using multivariate procedures, such as principal component (PC) analysis and clustering methods (k-means model). Four different ejaculate sub-populations were identified from two PCs that involved the head shape and size of the spermatozoa. The discriminant ability of the different morphometric sperm variables to predict sow litter size was analyzed using a receiver operating characteristics (ROC) curve analysis. Sperm head length, ellipticity, elongation, and regularity showed significant predictive capacity on litter size (0.59, 0.59, 0.60, and 0.56 area under curve (AUC), respectively). The morphometric sperm sub-populations were not related to sow litter size.

Knowledge of factors affecting semen quality could be of great importance for the collection and preservation of semen from threatened animals. To assess the effect of seasonality, sperm parameters and testosterone levels were examined throughout the year and compared with the distribution of conceptions. Cuvier's gazelle showed higher sperm quantity in April, coinciding with one peak of conceptions. In dorcas gazelle, sperm parameters showed a drop in October. However, percentage of conceptions increased during that month. In Mohor gazelle, sperm quality was best in April and August, in agreement with higher conception rates and high testosterone levels. Percentage of conceptions was correlated with photoperiod and rainfall in Cuvier's gazelle and with temperature in Mohor gazelle. To assess the effect of social environment, semen quality, testosterone and cortisol levels were quantified in males housed alone, in bachelor groups or with females. No differences were seen in Cuvier's and Mohor gazelles' semen traits, whereas dorcas males housed with females showed lower semen quality than males kept alone or with other males. Overall, ejaculate quality is influenced by seasonal factors in the three gazelle species, while social factors only appear to affect that of dorcas gazelle.

There has been very limited use of computer assisted semen analysis (CASA) to evaluate reptile sperm. The aim of this study was to examine sperm kinematic variables in American crocodile (Crocodylus acutus) semen samples and to assess whether sperm subpopulations could be characterized. Eight ejaculates (two ejaculates/male) from four sexually mature captive crocodiles were obtained. An ISAS®v1 CASA-Mot system, with an image acquisition rate of 50 Hz, and ISAS®D4C20 counting chambers were used for sperm analyses. The percentages of motile and progressively motile spermatozoa did not differ among animals (P > 0.05) but there was a significant animal effect with regards to kinematic variables (P < 0.05). Principal component (PC) analysis revealed that kinematic variables grouped into three components: PC1, related to velocity; PC2 to progressiveness and PC3 to oscillation. Subpopulation structure analysis identified four groups (P < 0.05), which represented, on average, 9.8%, 32.1%, 26.8%, and 31.3% of the total sperm population. Males differed in the proportion of sperm in each of the kinematic subpopulations. This new approach for the analysis of reptile sperm kinematic subpopulations, reflecting quantifiable parameters generated by CASA system technology, opens up possibilities for future assessments of crocodile sperm and will be useful in the future development of assisted reproduction for these species., Competing Interests: The authors have declared that no competing interests exist.

Much can be gained from the comprehensive study of a biological system. Based on what is known as Mayr's proximate-ultimate causation and the subsequent expansion to Tinbergen's four questions, biological traits can be understood by taking into account different approximations that try to explain mechanisms, development, adaptive significance or phylogeny. These, in principle, separate areas, can be integrated crossing boundaries, but bearing in mind that answers to one question would not explain a different query. Studies of sperm biology have, until now, not benefited much from this framework and potential integration. Proximate causes (particularly mechanisms) have been the subject of interest for reproductive biologists, and evolutionary explanations have been the domain of behavioural ecologists with interest in adaptive significance of traits in the context of post-copulatory sexual selection. This review will summarize opportunities for research in the different areas, focusing on sperm preparation for fertilization and suggesting possible integration within and between proximate and evolutionary studies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Spermatozoa are diverse in form and function and these differences impact on their fertilizing capacity. Because of considerable inter-male and inter-species differences in sperm traits, assessments of sperm quality demand that we consider variations at different levels. We should thus pay attention not only to average values but also intra- and inter-sperm population variations and subpopulation structure. Sperm shape and size evolve in reponse to postcopulatory sexual selection. Assessments of morphological variation, with conventional microscopy or with computer-assisted systems, should bear this in mind. In rodents sperm head shape is asymmetric so it requires more complex tools, such as geometric morphometrics. Sperm function also evolves under postcopulatory sexual selection and this could be used as a basis to assess sperm performance. Sperm cells swim actively to overcome barriers in the female tract and develop a peculiar motility pattern in the final stages prior to and during fertilization. Both types of movement can be analyzed by computer-assisted microscopy systems. Sperm have high energetic demands for cell homeostasis, motility, and signalling. Bioenergetics can be analyzed by various means, including extracellular flux analyses to characterize glycolysis and mitochondrial respiration. Finally, cell signalling during capacitation has received much attention and can be assessed by microscopy (conventional or computer-assisted) or flow cytometry. Recent advances in image-flow cytometry affords analyses of high cell numbers with spatial localization of subcellular changes, which will have a big impact in the development of functional tests for the andrology clinic and in sperm preservation and use in artificial insemination., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The ejaculate is heterogenous and sperm sub-populations with different kinematic patterns can be identified in various species. Nevertheless, although these sub-populations are statistically well defined, the statistical differences are not always relevant. The aim of the present study was to characterize kinematic sub-populations in sperm from two bovine species, and diluted with different commercial extenders, and to determine the statistical relevance of sub-populations through Bayesian analysis. Semen from 10 bulls was evaluated after thawing. An ISAS ® v1 computer-assisted sperm analysis (CASA)-Mot system was employed with an image acquisition rate of 50 Hz and ISAS ® D4C20 counting chambers. Sub-populations of motile spermatozoa were characterized using multivariate procedures such as principal components (PCs) analysis and clustering methods ( k-means model). Four different sperm sub-populations were identified from three PCs that involved progressiveness, velocity, and cell undulatory movement. The proportions of the different sperm sub-populations varied with the extender used and in the two species. Despite a statistical difference ( p < 0.05) between extenders, the Bayesian analysis confirmed that only one of them (Triladyl ® ) presented relevant differences in kinematic patterns when compared with Tris-EY and OptiXcell ® . Extenders differed in the proportion of sperm cells in each of the kinematic sub-populations. Similar patterns were identified in Bos taurus and Bos indicus . Bayesian results indicate that sub-populations SP 1 , SP 2 , and SP 3 were different for PC criteria and these differences were relevant. For velocity, linearity, and progressiveness, the SP 4 did not show a relevant difference regarding the other sperm sub-populations. The classical approach of clustering or sperm subpopulation thus may not have a direct biological meaning. Therefore, the biological relevance of sperm sub-populations needs to be reevaluated.

Background: Cysteine-RIch Secretory Proteins (CRISP) are expressed in the reproductive tract of mammalian males and are involved in fertilization and related processes. Due to their important role in sperm performance and sperm-egg interaction, these genes are likely to be exposed to strong selective pressures, including postcopulatory sexual selection and/or male-female coevolution. We here perform a comparative evolutionary analysis of Crisp genes in mammals. Currently, the nomenclature of CRISP genes is confusing, as a consequence of discrepancies between assignments of orthologs, particularly due to numbering of CRISP genes. This may generate problems when performing comparative evolutionary analyses of mammalian clades and species. To avoid such problems, we first carried out a study of possible orthologous relationships and putative origins of the known CRISP gene sequences. Furthermore, and with the aim to facilitate analyses, we here propose a different nomenclature for CRISP genes (EVAC1-4, "EVolutionarily-analyzed CRISP") to be used in an evolutionary context., Results: We found differing selective pressures among Crisp genes. CRISP1/4 (EVAC1) and CRISP2 (EVAC2) orthologs are found across eutherian mammals and seem to be conserved in general, but show signs of positive selection in primate CRISP1/4 (EVAC1). Rodent Crisp1 (Evac3a) seems to evolve under a comparatively more relaxed constraint with positive selection on codon sites. Finally, murine Crisp3 (Evac4), which appears to be specific to the genus Mus, shows signs of possible positive selection. We further provide evidence for sexual selection on the sequence of one of these genes (Crisp1/4) that, unlike others, is thought to be exclusively expressed in male reproductive tissues., Conclusions: We found differing selective pressures among CRISP genes and sexual selection as a contributing factor in CRISP1/4 gene sequence evolution. Our evolutionary analysis of this unique set of genes contributes to a better understanding of Crisp function in particular and the influence of sexual selection on reproductive mechanisms in general.

Sperm differentiation encompasses a complex sequence of morphological changes that takes place in the seminiferous epithelium. In this process, haploid round spermatids undergo substantial structural and functional alterations, resulting in highly polarized sperm. Hallmark changes during the differentiation process include the formation of new organelles, chromatin condensation and nuclear shaping, elimination of residual cytoplasm, and assembly of the sperm flagella. To achieve these transformations, spermatids have unique mechanisms for protein trafficking that operate in a coordinated fashion. Microtubules and filaments of actin are the main tracks used to facilitate the transport mechanisms, assisted by motor and non-motor proteins, for delivery of vesicular and non-vesicular cargos to specific sites. This review integrates recent findings regarding the role of protein trafficking in sperm differentiation. Although a complete characterization of the interactome of proteins involved in these temporal and spatial processes is not yet known, we propose a model based on the current literature as a framework for future investigations., Competing Interests: The authors declare no conflicts of interest.

Sperm competition is a powerful selective force that has influenced many reproductive traits in males and females although additional evolutionary explanations may help to understand the diversity of mammalian reproduction. Sperm morphology varies considerably in mammals with extreme examples in several rodent lineages in which a wide range of sizes and complex head morphologies have been identified. Mammalian spermatozoa also differ in function, with swimming velocity and trajectory showing much divergence. Underlying processes mediating function have received little attention so far, but differences in timing and proportion of sperm undergoing capacitation or acrosomal exocytosis may be related to variation in signalling processes. Furthermore, energy required for sperm functions (such as motion, signalling and overall maintenance of cell integrity) can be produced and consumed, following different patterns among species and this could be the result of several selective forces. A more thorough understanding of the diversity in structure and function of sperm cells, and underlying selective forces, may help us develop better methods to assess them taking into account particulars and generalities of sperm form and performance. Such tests could then become more reliable in estimations of the impact of cryopreservation or effect of changes in the environment and their relevance for fertility., (© 2019 Blackwell Verlag GmbH.)

ATP supply is essential for sperm performance and increases in ATP content coevolve with enhanced sperm swimming velocity as a response to sperm competition in rodents. ATP content is the balance between production and consumption but, although ATP production has received much attention, little is known about ATP consumption. The rate of ATP consumption is crucial for the propagation of the flagellar wave, becoming a main determinant of the time and distance sperm could move before exhausting their reserves. A high yield in distance per unit of ATP consumed (efficiency) could provide advantages in sperm competition. We characterized sperm ATP consumption rate in a group of mouse species with different sperm competition levels to understand its impact on swimming velocity, duration, and yield of sperm ATP reserves. Interspecific comparisons revealed that sperm of species with higher sperm competition levels had high ATP consumption rates and faster swimming velocity. Moreover, sperm that consumed ATP at a faster rate swam more efficiently, since they were able to cover more distance per unit of ATP consumed. Our results suggest that by coupling the advantages of higher ATP turnover rates to increased efficiency of ATP expenditure, sperm would respond to increasingly competitive environments while maintaining a positive ATP balance., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Whereas a broad link exists between nucleotide substitutions in the mitochondrial genome (mtDNA) and a range of metabolic pathologies, exploration of the effect of specific mtDNA genotypes is on-going. Mitochondrial DNA mutations are of particular relevance for reproductive traits, since they are expected to have profound effects on male specific processes as a result of the strict maternal inheritance of mtDNA. Sperm motility is crucially dependent on ATP in most systems studied. However, the importance of mitochondrial function in the production of the ATP necessary for sperm function remains uncertain. In this study, we test the effect of mtDNA polymorphisms upon mouse sperm performance and bioenergetics by using five conplastic inbred strains that share the same nuclear background while differing in their mitochondrial genomes. We found that, while genetic polymorphisms across distinct mtDNA haplotypes are associated with modification in sperm progressive velocity, this effect is not related to ATP production. Furthermore, there is no association between the number of mtDNA polymorphisms and either (a) the magnitude of sperm performance decrease, or (b) performance response to specific inhibition of the main sperm metabolic pathways. The observed variability between strains may be explained in terms of additive effects of single nucleotide substitutions on mtDNA coding sequences, which have been stabilized through genetic drift in the different laboratory strains. Alternatively, the decreased sperm performance might have arisen from the disruption of the nuclear DNA/mtDNA interactions that have coevolved during the radiation of Mus musculus subspecies., (© 2017 Society for Reproduction and Fertility.)

Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)