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ProAKAP4, AKAP4 precursor, is a specific protein from spermatozoa flagella, essential for maintaining motility efficiency. In all mammals studied proAKAP4 levels have been shown to correlate with total and progressive motility. In stallions and donkeys, proAKAP4 serves as an indicator of sperm quality and sustained motility over the time once thawed. This study aimed to compare proAKAP4 concentration with cytometry parameters: acrosome integrity, ROS production, mitochondrial potential and lipidic disorder. Animal cohort consisted of eight Asturcon ponies. Three ejaculates of each pony (24 ejaculates) were collected and frozen by commercial protocol. Post-thawed semen was analyzed by CASA and flow cytometry (PNA, JC1, HE, Fluo3 and M540 stains) and the proAKAP4 amount was quantified by ELISA commercial kit, Horse 4MIDⓇ Kit. Results show a positive correlation between proAKAP4 concentration and flow cytometry dyes: PNA stain (r= 0.34, p=0.0178), indicating membrane integrity; a negative correlation with membrane lipid disorder, M540 stain (r= -0.327, p=0.0234); a positive correlation with ROS production, H2 stain intensity (r= 0.48, p=0.005); and a negative correlation with mitochondrial potential measured by JC-1 (r=-0.41, p=0.0042). These results suggest that a spermatozoon with an intact membrane possesses more proAKAP4. Additionally, a spermatozoon with a high proAKAP4 concentration and an intact membrane also implies higher motility, resulting in increased ROS production. Conversely, a spermatozoon exhibiting significant lipid disorder shows a lower proAKAP4 concentration, indicating lower quality and reduced likelihood of reaching the fertilization site. As observed in previous publications, mitochondrial activity is not associated with proAKAP4 concentration, suggesting that the energy expenditure of the spermatozoon is not linked to the quality of movement defined by proAKAP4 concentration. A quality threshold of 42.10 ng of proAKAP4/10M of spermatozoa was calculated using a cluster between viability and motility parameters. This concentration is consistent with the 37.77 ng/10M of spermatozoa identified in a previous study, it may serve as a criterion for determining the sperm quality in post-thaw conditions. A relationship with cytometry parameters is observed showing that proAKAP4 is positively correlated with intact acrosome membrane and production of ROS which it suggests that an intact and rapid spermatozoon will show more proAKAP4 concentration. On the contrary, proAKAP4 is negatively correlate with a lipid disorder of the membrane which it means that a less quality spermatozoa will show less proAKAP4 concentration. Even if correlations are not high, their significance is strong, further analysis with a bigger cohort are needed to confirm these results. [ABSTRACT FROM AUTHOR]

The ever-evolving procedures of the practice of in vitro production (IVP) of embryos has well outpaced the ability of governments and institutions to create standardized rules and regulations around the global trade of these reproductive products. There are several challenges related to identifying and quantifying disease transmission risk in IVP embryos, not the least of which is a lack of published information. The International Embryo Technology Society (IETS) Health and Safety Advisory Committee (HASAC) has identified and addressed several of the challenges related to the potential for disease transmission via IVP embryos. This review will outline the current sanitation practices recommended by the IETS for in vivo derived (IVD) embryos, as well as the current practices at most in vitro fertilization (IVF) labs and will present the case that IVP embryos are not only just as safe as IVD embryos from a disease transmission risk standpoint, but perhaps may even be safer than IVD embryos, as the oocyte, zygote, and embryo in the in vitro production system has no contact with the downstream reproductive anatomy (fimbria, isthmus, oviduct, or uterus) of the donor female., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

ProAKAP4, a precursor of AKAP4 (A-kinase anchor protein) found in the flagellum of mammalian and non-mammalian spermatozoa, serves as a structural protein with established correlations to motility parameters across diverse species. This study aimed to determine the proAKAP4 level evolution in thawed stallion semen over a 3 h period, examining its correlation with motility descriptors and mitochondrial membrane potential. Utilizing sixteen ejaculates from four French warmblood stallions, this study involved maintaining thawed samples at 37 °C for 3 h, conducting proAKAP4 enzyme-linked immunosorbent assays (ELISA), computer-assisted sperm analysis (CASA), and mitochondrial membrane potential by JC-1 probe and flow cytometry at 0, 1, and 3 h post-thawing. The findings indicate significant positive correlations ( p ≤ 0.05) between proAKAP4 levels and sperm total or progressive motility at all time points analyzed. Spermatozoa velocity descriptors (VAP, VCL, VSL) and spermatozoa lateral head displacement (ALH) display positive correlations ( p ≤ 0.05) with ProAKAP4 at the 0 h post-thawing. ProAKAP4 concentration exhibits no discernible difference between batches with or without a cryoprotectant. Notably, proAKAP4 consumption remains insignificant within the initial hour after thawing but becomes significant ( p ≤ 0.05) between 1 and 3 h post-thawing. In summary, proAKAP4 demonstrates positive correlations with total and progressive motility in stallion semen for up to 3 h after thawing, albeit showing a noticeable decrease starting from the first hour post-thawing, indicating a progressive consumption as a result of spermatozoa motile activity.

ProAKAP4 is identified within the flagellum of spermatozoa in various mammalian species, serving as a structural protein associated with motility parameters. This investigation focuses on the presence of proAKAP4 in donkey sperm, elucidating its localization, molecular characteristics, and its correlation with motility descriptors and mitochondrial membrane potential. Twelve ejaculates from Catalan donkeys were analyzed in this study. The initial steps involved proAKAP4 sequencing and detection through Western blotting and immunofluorescence. Post-thaw assessments were conducted at 0, 1, and 3 h, encompassing proAKAP4 levels, sperm motility analyzed via Computer-Assisted Sperm Analysis (CASA), and mitochondrial membrane potential determined by flow cytometry using the JC-1 stain. The findings reveal that proAKAP4 in donkeys exhibits a characteristic localization at the principal piece of the flagellum, consistent with observations in other mammals. The molecular weight of proAKAP4 is determined to be 100 kDa. Significantly, a positive correlation (p ≤ 0.05) is established between proAKAP4 concentration and both total and progressive motility. The presence of cryoprotectant is associated with a lower proAKAP4 concentration. Notably, proAKAP4 experiences a substantial decrease (p ≤ 0.05) during the initial hour post-thawing. In conclusion, proAKAP4 is identified in donkey sperm, akin to its presence in other mammals. It exhibits a positive correlation with total and progressive motility, its concentration is notably affected by the presence of cryoprotectant with significant consumption observed during the initial hour following thawing. These findings contribute to our understanding of proAKAP4 dynamics in donkey sperm, providing insights that may have implications for semen preservation and reproductive technologies in equids., Competing Interests: Declaration of Competing Interest Maryse Delehedde and Nicolas Sergeant, co-authors of this manuscript, are cofounders of SPQI., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06−35.61 ng/10M spz; n = 23) or high (39.92−82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen.

Spermatozoa are highly differentiated cells whose ultimate function is fertilization to successfully transfer the male genome. This achievement relies on the expression, localization, organization, and proper functionality of their molecular components. For years, proteomics emerged as a remarkable approach for fertility research to identify specific protein markers related to sperm competency. Such biomarkers, undetected with conventional semen analysis methods, are next-generation tools to assess sperm functionality and predict male fertility. Among them, the proAKAP4 marker is a sperm-specific protein, synthesized as the precursor of AKAP4, highly conserved among mammals and only present in the principal piece of the flagellum. ProAKAP4 entails a disposable stock of AKAP4 to ensure long-lasting sperm motility up to the fecundation site, capacitation, and fertility. By being either converted into mature and active AKAP4 or degraded by proteolysis, proAKAP4 is a stress sensor, acting as a potential gatekeeper to prevent transmission of unfavorable genetic damage to the next generation. Loss or decrease of AKAP4 expression does not affect the number of spermatozoa produced but it impacted sperm motility, viability, and fecundation. A method based on proAKAP4 concentration measurement in a defined number of spermatozoa recently appeared as a simple, reliable, reproducible, robust, and quantitative tool to assess more objectively semen quality and predict male fertility. In this review, we will discuss how fundamental discoveries around the proAKAP4 biomarker provide a yet missing molecular dimension in semen analysis assessments to ensure higher semen quality and reproductive performance in veterinary clinics, zoos, and wild animal reproduction centers., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Functional sperm quality markers to predict bull fertility have been actively investigated. Among them, proAKAP4, which is the precursor of AKAP4, the main structural protein in the fibrous sheath of spermatozoa; appears to be promising, especially since spermatozoa lacking AKAP4 expression were shown to be immotile, abnormal, and infertile. In this study, the objective was to evaluate proAKAP4 concentration values with the classic sperm motility descriptors and fertility outcomes (NRR at 90 days) in post-thawed conditions of 10 bulls' semen. ProAKAP4 expression was confirmed by Western blotting and proAKAP4 concentrations were determined by ELISA. Variations in proAKAP4 concentrations were observed independently of the motility sperm descriptors measured using computer-assisted semen analysis (CASA). A ProAKAP4 concentration of 38.67 ± 8.55 ng/10 million spermatozoa was obtained as a statistical mean of all samples. Threshold values of proAKAP4 were then determined between 19.96 to 96.95 ng/10 million spermatozoa. ProAKAP4 concentrations were positively correlated with progressive motility and the linearity coefficient. The sperm showing the lowest progressive motility were the samples exhibiting proAKAP4 concentrations below 20 ng/10 million spermatozoa. Furthermore, proAKAP4 concentrations were significantly higher in bulls with a higher NRR in the field. Our results demonstrate a correlation between the semen concentration of proAKAP4 and NRR-90d ( p = 0.05) in post-thawed bull semen, highlighting the potential of proAKAP4 as a predictive marker of bull fertility.

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low-density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer-assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo-osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC-Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP-based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen., (© 2019 Blackwell Verlag GmbH.)