Semen Quality Research Articles

Deep learning classification method for boar sperm morphology analysis.

Boar semen quality emphasizes three major criteria: sperm concentration, motility, and morphology. Methods to analyze concentration and motility quickly and objectively readily exist, but few exist for analyzing morphology outside of subjective manual counting. Other vital factors for fertilization, like acrosome health, lack efficient detection methods due to limitations in detection by the human eye and costly biomarker analysis, which is rarely used in semen diagnostics. To overcome these challenges, we propose a novel approach integrating deep-learning technology with high-throughput image-based flow cytometry (IBFC) for objective and accurate analysis of both morphology and label-free acrosome health of thousands of individual spermatozoa at once, as opposed to manually counting on a microscope slide. Images of 10,000 spermatozoa were captured using an IBFC and manually annotated based on the primary morphological defect or acrosome health status for the training of the convolutional neural network (CNN). The CNN used these images to train and then applied that training to unannotated images to predict the model accuracy. Using the CNNs, high F1 scores of 96.73%, 98.55%, and 99.31% for 20x, 40x, and 60x magnifications, respectively, for morphological classification were attained. Additionally, the model demonstrates an F1 score of 99.8% in detecting subtle acrosome health variations at the 60x magnification. We have established an integrated approach to rapidly collect and classify morphological defects and acrosome health status, without the use of manual counting or biomarker labeling. Our study underscores the potential of artificial intelligence in semen diagnostics, reducing technician variability, streamlining assays, and facilitating the development of additional label-free detection methods. This innovative approach addresses the barriers hindering biomarker adoption in semen analysis, offering a promising avenue for enhancing reproductive health assessments.

Biostimulation methods based on chemical communication improve semen quality in male breeder rabbits

Biostimulation aims to optimize reproductive parameters as part of animal management practices by modulating animal sensory systems. Chemical signals, mostly known as pheromones, have a great potential in this regard. This study was conducted to determine the influence of short-term male rabbit exposure to different biological secretions, potentially pheromone-mediated, on reproductive parameters of males. Four groups of 18 males each were exposed to A) doe urine, B) 2-phenoxyethanol, C) doe vaginal swab, and D) distilled water (control), three times over a 2.5h exposure window, just before semen collection. Semen volume, sperm concentration and motility, as well as subpopulation analysis of the spermatozoa were assessed for each condition. Additionally, testosterone levels in blood samples were monitored at five time points over the 2.5 h exposure window. We found a higher percentage of motile, progressive, fast progressive and mid-progressive spermatozoa in any of the three experimental groups compared to the control group. In contrast, the semen volume and the percentage of immotile and non-progressive spermatozoa was generally higher in the control group. We then identified a higher proportion of a subpopulation of fast and progressive spermatozoa in groups A, B, and C compared to group D. Our data indicates that sperm motility increases when animals are exposed to specific biological fluids potentially containing pheromones, and that an increase in sperm volume does not correlate with an increase in spermatozoa concentration, progressiveness, and speed. Finally, no differences in testosterone levels were found among comparisons, although males of groups A and C (exposed to natural female biological fluids) showed a tendency towards higher testosterone levels. In conclusion, our results indicate that rabbit sperm quality increases upon exposure to the biological secretions proposed, thereby supporting further investigation into their molecular identity. This exploration could eventually pave the way for implementing the use of pheromones in rabbit husbandry to enhance reproductive and productive parameters in farmed rabbits.

PSXIII-16 Kinematics and viability of spermatozoa from Criollo and Angus bulls in a low-input system

Abstract The Chihuahuan Criollo cattle is a genetic resource with special adaptive characteristics to rough, arid and semi-arid ecosystems. There is a great interest in incorporating such adaptations into beef production systems as one of the strategies for sustainable production to face the climate change challenge. Reproductive success is determinant to achieve sustainability in animal production systems. Most research efforts to improve reproductive efficiency have focused on the study of females. However, it has been shown that much of the economic losses caused by embryonic deaths are due to the bull. The aim of this study was to evaluate the quality of thawed semen from Angus and Criollo bulls kept in a low-input system. Thirteen healthy bulls (n = 7 Angus and 6 = Chihuahuan Criollo), with an average age of 4.3 ± 0.48 yr, grazing in the same rangeland and subjected to natural pasture conditions, were studied. Semen obtained by electroejaculation, were frozen and subsequently thawed for analyses. Characteristics of motility, vitality (V), morphology (M) and plasma membrane integrity (PMI) were evaluated in fresh and thawed semen, whereas movement kinetics (MK) and acrosome reaction (AR) were evaluated in thawed semen. Statistical analysis was performed using the PROC GLM of SAS Studio 2022 including the fixed effects of breed, semen condition and their interaction for variables of progressive motility (PM), V, M, PMI and the effect of breed for variables of MK and AR. As expected, PM and V were less (P < 0.05) in thawed (57.93 ± 4.1 and 53.69 ± 2.7) compared with fresh (72.64 ± 3.9 and 74.98 ± 2.6) semen, whereas no differences (P > 0.05) were found in M. However, the interaction between semen condition and breed for PMI tended to the significance (P = 0.08) with a PMI of 68.95 ± 4.6 and 58.98 ± 4.3 for Criollo and Angus, respectively, in fresh semen, whereas in thawed semen this difference was inverted (52.08 ± 4.6 and 58.40 ± 4.6). It is important to note that the semen retained quality traits with values above the minimum accepted for use in artificial insemination, despite the scarcity of resources of the system in which the animals were kept. There were no differences by breed in the main MK variables; however, AR reported as live unreacted sperm was less (P < 0.05) in Criollo (40.49 ± 4.49) compared with Angus bulls (58.48 ± 4.49). In conclusion, it is important to incorporate the assessment of membrane integrity and acrosome reaction in thawed semen evaluation to obtain a more accurate prediction of the potential fertility of samples, even if motility parameters appear to be optimal.

PSIII-6 Multi-sire artificial insemination of beef heifers in the Nebraska Sandhills effect on pregnancy rate compared with single-sire insemination

Abstract The objective of this study was to compare pregnancy outcomes resulting from artificial insemination (AI) with multi-sire semen (MS) compared with single-sire semen (SS) in beef heifers. Another study observed pregnancy and paternity outcomes to sexed MS without a control treatment. In the first study, unrelated ABS Global Angus bulls (n = 3) were selected for AI to 441 (Yr 1) and 391 (Yr 2) Angus crossbred heifers. Heifers were synchronized with the melengestrol acetate – prostaglandin F2α timed-AI protocol. Conventional semen, from each bull, were labeled A, B, or C. Remaining semen was pooled in equal amounts to form conventional MS straws. Heifers were AI using alternating treatment of MS or SS: A, B, or C with one-half of inseminated heifers receiving either MS or SS. The GLIMMIX procedure in SAS 9.4 analyzed responses to treatments using the BIN distribution option and reported as LSMEANS. Contrasts and estimates analyzed responses between treatments. Ultrasound determined SS pregnancy rate averaged 60% (SS-A = 64%, SS-B = 58%, SS-C = 58%) and MS average 62% (P = 0.63). Paternity was determined in 57 MS calves and confirmed in 49 SS calves. In Yr 1, Bull A sired 7%, bull B sired 56%, and bull C sired 37%. Although bull A was much less successful than bull B or C, the values are not significantly different due to the small sample size (P = 0.11 and P = 0.24, respectively). Few conclusions can be made from the ongoing study, but it is hypothesized MS may improve consistency of pregnancy rate by equilibrating semen quality in herds subjected to timed AI. In the observational study, multiple 5-way cross bulls born from the ranch herd were selected for AI of 951 ± 41 heifers for 3 yr. Heifers were synchronized using the previously mentioned protocol with split-time AI. Unequal amounts of semen were collected, pooled, and sexed to favor heifer progeny at an expected ratio of 9:1. Semen from 2 to 4 random bulls were pooled making 3 to 4 MS groups each year. Heifers exhibiting estrus were administered sexed MS, but there was no control treatment. The observed ultrasound determined average pregnancy rate of heifers administered sexed MS was 65% (Yr 1), 75% (Yr 2), and 72% (Yr 3). Previous literature suggests average pregnancy rate to AI with sexed SS is 53%. Paternity testing in Yr 2 found semen contribution mirrored paternity ratios within some groups but the sire group with 2 bulls found one sired 15% and the other sired 85%. This study exemplified a viable method for AI that requires further research. Further research into semen quality of a single sire, and pooled semen could lead to optimization of fertilization among individuals within a herd.

PSIII-1 The effects of maternal diet on ram lamb reproductive characteristics

Abstract Maternal nutrition during gestation and resulting impacts on ram reproduction and epigenetics are poorly understood. We previously reported altered mRNA and epigenetic markers in F1 ram sperm due to poor maternal diet (restricted- and over-feeding) during gestation, yet the multigenerational effects on ram reproduction are unexplored. We hypothesized that F2 offspring born from restricted- and over-fed granddams would exhibit altered reproductive characteristics, and F1 and F2 offspring would display altered circulating concentrations of reproductive hormones. The objectives of this study were to determine the effects of poor maternal diet on 1) the reproductive function of F2 ram offspring, and 2) key reproductive hormones in F1 and F2 offspring. To evaluate these objectives, multiparous Dorset ewes (F0, n = 46) were fed 100% (CON), 60% (RES), or 140% (OVER) of National Research Council requirements from d 30 of gestation through parturition and a control diet during lactation. Offspring are identified as CON-F1 (n = 10 ewes; 12 rams), RES-F1 (n = 13 ewes; 21 rams), and OVER-F1 (n = 16 ewes; 15 rams). The F1 ewes (n = 37) were maintained on a control diet throughout and were bred between 16 and 19 mo of age to establish the F2 generation. Resulting male offspring are referred to as CON-F2 (n = 6 rams), RES-F2 (n = 13 rams), and OVER-F2 (n = 9 rams) in correspondence to the diets of the granddam. Semen samples of F2 rams were collected via electroejaculation at d 248.4 ± 0.36 (collection 1) and d 255.4 ± 0.36 (collection 2). Semen quality (volume, pH, sperm motility, and sperm concentration) was evaluated at both collections using mCASA; iSperm, Aidmics Biotechnology Co., LTD. Scrotal circumference was measured at collection 2. Sperm morphology was determined by contrast microscopy. Testosterone (T) and follicle stimulating hormone (FSH) were measured in F1 and F2 serum samples collected at d 256 ± 0.13 using commercially available kits. Data were analyzed using R Studio with P ≤ 0.05 considered significant and 0.05 < P ≤ 0.10 considered a tendency. An effect of granddam diet was not observed for F2 ram semen volume, pH, sperm motility, sperm concentration, scrotal circumference, nor histological sperm morphology (P ≥ 0.25). An effect of F0 diet was not observed for F1 or F2 offspring serum concentrations of T and FSH (P ≥ 0.28). In our model, F0 diet during gestation did not affect F2 male offspring sperm quality or circulating T and FSH in F1 and F2 generations. However, based on our previous reports that F0 diet altered F1 sperm epigenome, there may be an impact of maternal diet on epigenetic inheritance which warrants further exploration.

Role of Platelet-Rich Plasma in the Management of Non-obstructive Azoospermia.

Non-obstructive azoospermia (NOA) is a common cause of infertility in males, which is characterized by the absence of sperm in the ejaculate, resulting from impaired spermatogenesis. The primary therapeutic approaches for the management of NOA include testicular sperm extraction, varicocelectomy in case of clinical varicoceles, and hormonal manipulation. While traditional treatments are found to have a limited role in the management of NOA, recent studies have explored the potential of platelet-rich plasma (PRP) as a safer and promising therapeutic option. Autologous PRP preparations are derived from the patient's blood and comprise growth factors and cytokines, promoting tissue repair and regeneration. PRP has a wide range of applications in the medical field, including managing infertility in males and females. This literature review aimed to evaluate the existing evidence on the efficacy and safety of PRP in the management of NOA. After a thorough review of relevant data from observational and experimental studies, the findings of this study suggested that PRP may positively influence spermatogenesis, sperm quality, DNA integrity, and sperm retrieval during assisted reproductive procedures. Further research is needed to establish the optimal PRP preparation, administration method, and long-term benefits. The newer studies shall include a diverse patient population and employ long-term follow-up to assess the durability of any positive effects of PRP treatment. The growing body of evidence regarding the therapeutic potential for NOA in humans offers greater opportunities for men seeking fertility treatment, informing clinical practice, and optimizing the use of PRP.

PSVII-28 Unraveling the mechanisms governing boar semen quality during chilled storage

Abstract Artificial insemination (AI) appears as a core of reproductive management in the hog industry. AI involves the use of chilled semen doses meeting routine thresholds for motility (≥ 70%) and morphology (≥ 80%) at collection. However, semen doses meeting these standards exhibit a gradual decline in sperm quality, with likely divergent profiles during preservation that impact fertility outcomes. Hence, this study aims to unravel the sperm quality dynamics of boar semen doses during 7-d chilled storage at 17ºC. Semen doses (n = 25) from proven-fertile Duroc boars were provided by a commercial hog farm (Prestage Farm, MS) and transported to the laboratory within 30 min of collection. Each semen sample was aliquoted and analyzed on the day of collection (d 0) and after 7 d of storage (d 7) at 17°C. Sperm total motility (TM), progressive motility (PM), and normal morphology (NM) were assessed using a computer-assisted sperm analyzer (CASA; CEROS II, IMV Biotechnologies). Apoptosis (Annexin V/7-AAD), viability (Propidium iodide – PI), and intracellular reactive oxygen species (ROS; DCFH-DA) were evaluated using flow cytometry. Total antioxidant activity (TAOC) and lipid peroxidation (malondialdehyde; MDA) were assessed using commercial kits (Cayman Chemical). Data analysis was conducted using parametric statistics (SPSS), with P < 0.05 considered significant. Results were presented as mean ± SEM. Data analysis confirmed the significant decrease of CASA parameters, particularly TM from d 0 to d 7 (78.87 ± 1.37 vs. 73.19 ± 1.98%), with extreme variance on d 7 allowing to discriminate high motility (HM, n = 6) vs low motility (LM, n = 6) semen doses. HM doses evidenced significantly greater (P < 0.05) TM (83.73 ± 1.17%), PM (36.07 ± 1.11%), and NM (89.45 ± 2.30%) compared with LM doses (TM: 59.40 ± 3.35%, PM: 16.57 ± 3.25%, NM: 79.93 ± 2.61%) on d 7. Unlike HM samples, which were comparable between d 0 and d 7, LM doses displayed significantly greater proportions of apoptotic and viable cells, and increased intracellular ROS levels on d 0. Likewise, LM samples revealed significantly greater (P < 0.05) proportions of apoptotic cells (3.69 ± 1.66%), dead cells (38.41 ± 3.71%), and cells with intracellular ROS (36.21 ± 3.62%) than HM samples (apoptotic cells: 0.03 ± 0.02%, dead cells: 4.73 ± 4.65%, ROS: 5.34 ± 4.91%) on d 0; however, only the proportions of dead cells displayed significant increase in LM on d 7. MDA concentrations were comparable between HM and LM samples on d 0. However, on d 7, LM samples exhibited significantly greater MDA concentrations than HM samples. In contrast, TAOC was significantly greater in HM samples (2.53 ± 0.14 mM) compared with LM samples (2.17 ± 0.07 mM) on d 0. In conclusion, semen samples with similar gross characteristics at collection exhibited differential responses during storage. Their initial redox potential and apoptosis-like changes may contribute to their varying ability to withstand prolonged storage. Work supported by USDA-ARS grant# 6066-31000-015-00D.

PSV-15 Evaluating the relationship between subcutaneous backfat thickness and sperm morphology in young beef bulls

Abstract Literature indicates that high-energy diets can lead to reductions in both sperm motility and morphology in bulls. This study aimed to investigate the relationship between individual variation in subcutaneous backfat thickness (SCBF) and sperm morphology in young beef bulls exposed to the same diet. We hypothesized that bulls with excessive subcutaneous fat thickness have increased sperm morphology defects compared with bulls with adequate subcutaneous backfat thickness. Historical data collected from yearling Bos taurus beef bulls (n = 557) enrolled in a bull development program were used in an observational retrospective cohort study to test this hypothesis. Over the course of 5 yr, bulls in two locations were developed according to industry standard practices, and performance was evaluated over 84 d with a 21-d adaptation period. At the end of the performance evaluation period, a carcass ultrasound was performed to evaluate ribeye area, SCBF, and intramuscular fat. Furthermore, breeding soundness examinations (BSE) were performed according to the Society of Theriogenology guidelines within 40 d after carcass ultrasonography. Bulls that failed the BSE for reasons unrelated to semen quality were excluded from this study. Within each year, bulls were ranked based on SCBF and classified as top 10% (TOP; n = 55), middle 80% (MID; n = 446), and bottom 10% (BTM;n = 56) for SCBF. Initial body weight (BW) was greater (P < 0.01) in TOP bulls compared with MID and BTM (Table 1). Off-test BW was also greater in TOP compared with MID and BTM bulls (P < 0.01). In contrast, average daily gain and scrotal circumference did not differ between experimental groups (P = 0.35 and P = 0.65, respectively). TOP bulls had greater (P < 0.01) SCBF compared with MID and BTM, and MID bulls had greater (P < 0.01) SCBF compared with BTM bulls. Similar results were observed for intramuscular fat (P < 0.01) and ribeye area (P < 0.01). Percentage of normal sperm cells was greater in both BTM and MID bulls compared with TOP bulls (P < 0.01). Additionally, percentage of primary abnormalities tended (P = 0.07) to be greater in TOP bulls compared with BTM and MID. There were no differences in the percentage of secondary abnormalities between experimental groups (P = 0.45). There was a tendency (P = 0.07) for a greater percentage of TOP bulls to fail the BSE compared with MID and BTM bulls. Moreover, there was a positive linear relationship (P = 0.01) between SCBF and the probability of failing the BSE. In summary, elevated SCBF in bulls exposed to the same diet was associated with an increase in sperm morphological abnormalities and tended to increase the percentage of bulls failing the BSE.

A Comparison of White and Yellow Seminal Plasma Phosphoproteomes Obtained from Turkey (Meleagris gallopavo) Semen.

Seminal plasma is rich in proteins originating from various male reproductive organs. The phosphorylation of these proteins can significantly impact sperm motility, capacitation, and acrosome reaction. Phosphoproteomics identifies, catalogues, and characterizes phosphorylated proteins. The phosphoproteomic profiling of seminal plasma offers valuable insights into the molecular mechanisms that influence semen quality and male fertility. Thus, the aim of this study was a phosphoproteomic analysis of white and yellow turkey seminal plasma. The experimental material consisted of 100 ejaculates from BIG-6 turkeys between 39 and 42 weeks of age. The collected white and yellow turkey seminal plasmas were analyzed for total protein content; the activity of selected enzymes, i.e., alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT); and the content of reduced glutathione (GSH) and malondialdehyde (MDA). Phosphoproteins were isolated from white and yellow seminal fluids, and the resulting protein fractions were separated by SDS-PAGE and Western blotting. Phosphorylated residues were immunodetected, and the isolated phosphoproteins were identified (nano LC-MS/MS). Yellow seminal plasmas were characterized by higher levels of total protein, GSH, and MDA, as well as higher levels of ALP, ACP, and GPx activity. There were no significant differences in the activity of SOD and CAT. A total of 113 phosphoproteins were identified in turkey seminal fluids. The functional analysis demonstrated that these phosphoproteins were mainly involved in oocyte fertilization, organization and metabolism of the actin cytoskeleton, amplification of the intracellular signal transduction pathway, general regulation of transport, vesicular transport, proteome composition of individual cellular compartments, and the organization and localization of selected cellular components and macromolecules. Increased phosphorylation of the fractions containing proteins encoded by SPARC, PPIB, TRFE, QSOX1, PRDX1, PRDX6, and FASN genes in white plasmas and the proteins encoded by CKB, ORM2, APOA1, SSC5D, RAP1B, CDC42, FTH, and TTH genes in yellow plasmas was observed based on differences in the optical density of selected bands. The obtained results indicate that the phosphorylation profiles of turkey seminal plasma proteins vary depending on the type of ejaculate.

26 Trace minerals to improve bull fertility

Abstract Bull fertility is paramount for reproductive success in cow-calf production systems and there are a variety of factors that influence bull fertility. Trace mineral requirements and recommendations have been established for growing and finishing steers, as well as lactating and pregnant cows, yet no specific requirements have been established for young or mature beef bulls. Nonetheless, trace minerals have been shown to have an important role in sexual maturation and semen quality of ruminants. Research performed in rams indicates that zinc deficiency results in reduced testicular development, decreased testicular concentrations of testosterone, and impaired development of seminiferous tubules. Deficiency of Zn has also been reported to negatively impact male fertility in humans, where men diagnosed with infertility responded positively to Zn supplementation. In growing bulls, diets with greater concentrations of organic forms of Zn, Cu, and Mn, tended to result in earlier attainment of puberty compared with control diets containing inorganic minerals. Furthermore, greater liver concentrations of Zn have been negatively correlated with sperm acrosomal damage in growing bulls, based on flow cytometry analyses. More recent research indicates that the use of hydroxichloride-based trace minerals results in greater liver concentrations of Zn and Cu compared with sulfate sources. While evidence indicates that trace mineral status has an important role in bull fertility, further research is required to better understand the contributions of trace minerals to semen quality. In addition, investigating the impact of trace mineral sources with greater bioavailability on semen quality may result in the development of practical recommendations that optimize bull fertility.

Dehydrozingerone ameliorates arsenic-induced reproductive toxicity in male Wistar rats.

Arsenic (As3+), a significant environmental pollutant that has garnered global attention, is widely recognized for its adverse effects on reproductive health. This study assesses the aphrodisiac activity of Dehydrozingerone (DHZ) against As3+ induced sexual dysfunction in male Wistar rats. Male Wistar rats were divided into control, As3+, and As3++DHZ groups. The As3+ group received 5mg/kg sodium arsenite (NaAsO2) orally while As3++DHZ group received 50mg/kg synthesized DHZ along with As3+ for 42 days. Following administration, mount and intromission latency, frequency, and average time were measured to assess aphrodisiac and reproductive toxicity in male Wistar rats which had 1:1 coitus with female rats. On days 14th, 28th, and 42nd, sexual behaviour was measured. Further on 43rd day, animals were sacrificed, blood was collected to measure oxidative parameters and LH hormone, and then testes were collected to profile reproductive damage. As3+ treated rats had lower sperm counts, motility, and abnormalities. These alterations reduced sexual hormones. In addition, As3+ toxicity depleted antioxidant indicators including SOD, GSH and elevated ROS. Compared to the As3+ group, As3++DHZ showed a substantial (p < 0.05) increase in sperm count, motility, and reduced abnormalities. DHZ also reversed the rise in luteinizing hormone caused by As3+ therapy, restored oxidative indicators, and improved seminiferous tubule structural damage. 42 days As3+ exposure slightly increased rats' sexual desire but not sperm quality. However, As3++DHZ lower libido and sperm quality. Thus, DHZ therapy enhanced rat sexual desire and sperm quality compared to As3+.

Environmental arsenic exposure and reproductive system toxicity in male and female and mitigatory strategies: a review.

Environmental Arsenic (As) exposure is one of the main health challenges in different area of the world. As is a significant factor responsible to the reproductive system toxicity in both male and female. In this study, the most important effects mechanisms and biomarkers related to environmental exposure to As and the reproductive system toxicity, and infertility risk are reviewed in male and female. The results showed that the most important As-induced reproductive system toxicity in the male were alteration in the quantity and quality of semen, testicular toxicity, oxidative stress, testosterone reduction, and sperm apoptosis. For female were oxidative stress, spontaneous miscarriage, reproductive cycle disruption, decrease in the estradiol, progesterone, and testosterone levels and impair fecundity. The main mechanisms of reproductive system toxicity caused by As exposure in male were, genotoxic effects, reduction of glutathione, disruption of sex hormones, sperm flagellum formation impairment, inhibition of spermatogenesis, disruption of cell signaling pathways, and metabolites disruption. For female were abnormal signaling in gene expression, hormonal homeostasis, As-accumulation in placental tissue and creation of reactive oxygen, disruption in the neurotransmitters balance, and sex hormones disruption. The suitable biomarkers for As-induced reproductive toxicity in male were changes in testosterone, one-carbon and lipid metabolism, noncoding RNAs, and steroid hormone homeostasis, and for female was human chorionic gonadotropin (hCG) changes. Finaly, taking selenium, zinc, silymarin, vitamins (C and E) and phytonutrients can be effective in reducing the As-induced reproductive system toxicity and infertility risk.

Impact of human papillomavirus and coinfection with other sexually transmitted pathogens on male infertility.

This study primarily aimed to investigate the prevalence of human papillomavirus (HPV) and other common pathogens of sexually transmitted infections (STIs) in spermatozoa of infertile men and their effects on semen parameters. These pathogens included Ureaplasma urealyticum, Ureaplasma parvum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus 2, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus agalactiae, Pseudomonas aeruginosa, and Staphylococcus aureus. A total of 1951 men of infertile couples were recruited between 23 March 2023, and 17 May 2023, at the Department of Reproductive Medicine of The First People's Hospital of Yunnan Province (Kunming, China). Multiplex polymerase chain reaction and capillary electrophoresis were used for HPV genotyping. Polymerase chain reaction and electrophoresis were also used to detect the presence of other STIs. The overall prevalence of HPV infection was 12.4%. The top five prevalent HPV subtypes were types 56, 52, 43, 16, and 53 among those tested positive for HPV. Other common infections with high prevalence rates were Ureaplasma urealyticum (28.3%), Ureaplasma parvum (20.4%), and Enterococcus faecalis (9.5%). The prevalence rates of HPV coinfection with Ureaplasma urealyticum, Ureaplasma parvum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus 2, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus agalactiae, and Staphylococcus aureus were 24.8%, 25.4%, 10.6%, 6.4%, 2.4%, 7.9%, 5.9%, 0.9%, and 1.3%, respectively. The semen volume and total sperm count were greatly decreased by HPV infection alone. Coinfection with HPV and Ureaplasma urealyticum significantly reduced sperm motility and viability. Our study shows that coinfection with STIs is highly prevalent in the semen of infertile men and that coinfection with pathogens can seriously affect semen parameters, emphasizing the necessity of semen screening for STIs.

The experience of transfeminine adolescents and their parents regarding fertility preservation via testicular sperm extraction (TESE): a qualitative study.

How do transfeminine adolescents and their parents experience fertility preservation via testicular sperm extraction (TESE)? Participants experienced the TESE process positively when provided with appropriate guidance to navigate the decisional dilemma between preserving future biological parenthood and the pressure to start puberty suppression early. Sperm banking through ejaculation is not feasible for all transfeminine adolescents due to genital dysphoria and early puberty; for this group, TESE is the only alternative. However, during early puberty, they must postpone or pause puberty suppression until spermatogenesis is fully developed. All consecutive TESE patients in our centre and their parents were invited to participate. Between December 2022 and May 2023, we included 6 adolescents and 10 parents. We used a qualitative approach based on semi-structured interviews to study the experience of the transfeminine adolescents and their parents. Transfeminine adolescents diagnosed with gender dysphoria in early puberty (Tanner stage ≥2) who underwent TESE before the start of puberty suppression or gender-affirming hormones participated in this study. The interviews were recorded, manually transcribed and analysed using reflective thematic analysis focusing on understanding the participants' experiences of puberty and fertility preservation and the various factors and social processes influencing their decision to undergo TESE. Transfeminine adolescents decide to undergo invasive fertility preservation because of a possible, future desire for parenthood. Parents stressed the importance of preventing future decisional regret. They both saw masturbation to obtain a semen sample as unreachable. Even though TESE is a surgical procedure with possible complications and pain afterwards, this alternative seemed less dysphoric to them.Adolescents had to postpone the start of puberty suppression, waiting for spermatogenesis to complete. Given the unpredictable development of puberty, they felt a lot of stress and anxiety during this period and were primarily concerned about the lowering of their voice. Because of this puberty-related stress, they were constantly balancing their motivations for fertility preservation against the early start of puberty suppression to prevent unwanted irreversible pubertal body changes. To support this decisional dilemma, adolescents and their parents need adequate communication with each other and their healthcare providers to reflect on their stress for pubertal change. Furthermore, close, continuous surveillance of their current mental state by their parents, together with their psychologist and pubertal development by their physicians, is needed. Despite the challenging decision and postponement of puberty suppression, they retrospectively experienced it positively without any regret. All adolescents successfully underwent TESE, with viable spermatozoa obtained and without complications. This may result in a more positive reflective experience regarding fertility preservation. Furthermore, the perspectives of adolescents with the intention of TESE but who already started with puberty suppression before the preservation were not included. This study helps healthcare providers improve their counselling and guidance of transfeminine adolescents who consider postponing puberty suppression for fertility preservation via TESE. The adolescents and their parents know more clearly what to expect during fertility preservation. Specific peer-support programs may facilitate the decision process for upcoming adolescents. No external funding was used for this study. None of the authors have any conflicts of interest. Not applicable.

Telomeric RNAs, TERRA, as a Potential Biomarker for Spermatozoa Quality.

Characterization of long non-coding telomeric repeat-containing RNAs in sperm of normozoospermic and oligoasthenozoospermic men as new biomarker of idiopathic male infertility. We conducted an observational prospective study with two groups of men with normal or orligoasthenozoospermic spermiogram, aged 40 and above. Fertility parameters were analyzed in men undergoing intracytoplasmic sperm injection with donor oocytes, to avoid the female factor. Telomeric RNAs and telomere length were measured by quantitative fluorescent in situ hybridization. Data from seminal parameters and in-vitro fertilization were assessed according to IVIRMA protocols. Patients with oligoasthenozoospermia, who had worse seminal parameters, also obtained embryos with lower inner-cell-mass quality (p = 0.04), despite using donor oocytes. While mean levels of telomeric RNAs were similar for both groups, the percentage of spermatozoa with more than 3 foci was higher in oligoasthenozoospermic men (p = 0.02). Regarding telomere length, oligoasthenozoospermic men had shorter mean, a higher accumulation of short telomeres (15th percentile; p = 0.03) and a lower percentage of very-long telomeres (85th percentile; p = 0.01). Finally, a positive correlation was found between telomeric-RNAs intensity and total progressive motility in the spermatozoa of normozoospermic patients (r = 0.5; p = 0.03). Telomeric parameters were altered in the spermatozoa of the oligoasthenozoospermic group, which also showed lower quality embryos. Interestingly, in the normozoospermic group, a correlation was found between progressive motility and telomeric RNA levels, suggesting that they could be a good biomarker of sperm quality. Further studies are required to confirm these results and translate them into the clinical practice.Trial registration number: 1711-MAD-109-CB, 07/07/2021.

High temperature induces motility of zebrafish sperm after short-term storage

Short-term storage on ice of zebrafish (Danio rerio) sperm has been successfully applied in diverse basic experimental research. The optimal and economically efficient short-term storage method to date, however, can only maintain the fertilization ability of the sperm for a few hours. In this study, we aimed to improve zebrafish sperm quality after short-term storage and to test whether this affects the nucleotide cargo of spermatozoa. Zebrafish sperm was diluted with E400 extender (130 mM KCl, 50 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM D-(+)-Glucose and 30 mM HEPES-KOH, pH 7.9, 400 mOsmol/kg) at a dilution ratio 1:20 (v/v) in tubes (500 μL) and stored for 24 h at 0–2 °C under aerobic conditions. Elevating the temperature of sperm suspension at 28 °C for 20 min significantly enhanced sperm motility during activation in both distilled water and Perchec solution with 0.25 % Pluronic F-127. Despite the observed decline in sperm motility during short-term storage, Perchec solution (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8.0, 160 mOsmol/kg) enhanced activation of the aged sperm compared to activation of distilled water. Following 24 h of sperm storage, higher fertilization, and hatching rates were observed when sperm suspension was incubated at 28 °C for 20 min and activated in Perchec solution at a ratio of approximately 6120 spermatozoa per egg, compared to activation in hatchery water. A tentative RNA-seq study identified a set of 2180 differentially expressed genes after 24 h of sperm storage, significantly enriched in the glycosaminoglycan degradation and galactose metabolism pathways. The validity was discussed due to the limited mRNA content in spermatozoa. No alterations were identified in the DNA methylation level of 24 h stored sperm and neither differentially methylated CpGs (DMCs) nor differentially methylated regions (DMRs) were detected between fresh and aged sperm. Based on these phenotypic and molecular investigations and the successful implementation of high-temperature incubation prior to sperm activation in artificial reproduction, we propose that high-temperature incubation following short-term sperm storage at low temperatures is a safe and significant approach in zebrafish and holds potential benefits for other economically important warm-water species in aquaculture.

Assessment of male creatinine levels and fecundity in couples planning pregnancy: a national cohort study in China.

Is there an association between male creatinine levels and time to pregnancy (TTP) in couples planning pregnancy? Low and high male creatinine concentrations were associated with reduced couple fecundity. Abundant evidence suggests male creatinine dysfunction is associated with infertility in males with kidney diseases. However, the association of preconception creatinine levels with reduced fecundity among general reproductive-aged couples lacks evidence from an in-depth population study. Based on the population-based cohort study from the National Free Preconception Check-up Projects, 4023204 couples were recruited and met the inclusion criteria from 1 January 2015 to 31 December 2017. They were planning pregnancy and were followed up every 3 months until achieving pregnancy as detected by gynaecological ultrasonography or were followed up for 1 year for the analysis of TTP. Cox regression models were used to estimate hazard ratios (HRs) and 95% CI for creatinine deciles. Restricted cubic spline regression was adopted for the dose-response relationship of creatinine with HRs. R statistical software was used for data analysis. Of the included participants, 2756538 (68.52%) couples successfully conceived. The median male serum creatinine was 81.50 μmol/l. Compared with the reference group (78.00-81.49 μmol/l) including the median creatinine, fecundity in the first (≤64.89 μmol/l), second (64.90-69.99 μmol/l), third (70.00-73.99 μmol/l), and tenth (≥101.00 μmol/l) deciles decreased by 8%, 5%, 2%, and 1%, respectively (Decile 1 Adjusted HR 0.92, 95% CI 0.91-0.92; Decile 2 Adjusted HR 0.95, 95% CI 0.95-0.96; Decile 3 Adjusted HR 0.98, 95% CI 0.97-0.99; Decile 10 Adjusted HR 0.99, 95% CI 0.98-0.99). An inverse-U-shaped association was consistently presented among males such that non-inferiority for fecundity was shown when creatinine was in the 81.66-104.90 μmol/l range (P for non-linearity < 0.001). For males over 40 years old, the risk of fecundity impairment was more obvious and the recommended range of creatinine levels for TTP was reduced and more narrow, compared with that for younger males. Not including the time couples spend preparing for pregnancy before enrolment would lead to an overestimation of fecundity; additionally some couples place pregnancy plans on hold due to special emergencies, which would not have been recognized. Due to the lack of information regarding semen quality, psychological factors, sexual intercourse frequencies, and hazardous environmental factors, we could not adjust for these factors. Some variates were self-reported and dichotomized, which were prone to bias. Direct variables reflecting muscle mass and impaired kidney function were lacking. Thus, extrapolation should be done with caution. Male creatinine is associated with couples' fecundity and the relationship varied by age. This study provides a better understanding of the potential implications and significance of different creatinine levels and their association with the clinical significance regarding couples' fecundity. This research has received funding from the National Natural Science Foundation of China (Grant No. 81872634), the Basic Research Funds of Central Public Welfare Research Institutes of China (Grant No. 2023GJZ03), the National Key Research and Development Program of China (Grant No. 2016YFC1000307), and the Project of National Research Institute for Family Planning (Grant No. 2018NRIFPJ03), People's Republic of China. The authors have no conflicts of interest to declare. N/A.

Whole exome sequencing analysis of 167 men with primary infertility

BackgroundSpermatogenic failure is one of the leading causes of male infertility and its genetic etiology has not yet been fully understood.MethodsThe study screened a cohort of patients (n = 167) with primary male infertility in contrast to 210 normally fertile men using whole exome sequencing (WES). The expression analysis of the candidate genes based on public single cell sequencing data was performed using the R language Seurat package.ResultsNo pathogenic copy number variations (CNVs) related to male infertility were identified using the the GATK-gCNV tool. Accordingly, variants of 17 known causative (five X-linked and twelve autosomal) genes, including ACTRT1, ADAD2, AR, BCORL1, CFAP47, CFAP54, DNAH17, DNAH6, DNAH7, DNAH8, DNAH9, FSIP2, MSH4, SLC9C1, TDRD9, TTC21A, and WNK3, were identified in 23 patients. Variants of 12 candidate (seven X-linked and five autosomal) genes were identified, among which CHTF18, DDB1, DNAH12, FANCB, GALNT3, OPHN1, SCML2, UPF3A, and ZMYM3 had altered fertility and semen characteristics in previously described knockout mouse models, whereas MAGEC1,RBMXL3, and ZNF185 were recurrently detected in patients with male factor infertility. The human testis single cell-sequencing database reveals that CHTF18, DDB1 and MAGEC1 are preferentially expressed in spermatogonial stem cells. DNAH12 and GALNT3 are found primarily in spermatocytes and early spermatids. UPF3A is present at a high level throughout spermatogenesis except in elongating spermatids. The testicular expression profiles of these candidate genes underlie their potential roles in spermatogenesis and the pathogenesis of male infertility.ConclusionWES is an effective tool in the genetic diagnosis of primary male infertility. Our findings provide useful information on precise treatment, genetic counseling, and birth defect prevention for male factor infertility.

THE EFFECTIVENESS OF VITAMIN C AND E ADDITION IN VARIOUS DILUENTS OF BRAHMAN CATTLE SPERMATOZOA AFTER FREEZING

This research aimed to determine the quality of Brahman cattle spermatozoa after freezing in various diluent ingredients (Egg Yolk Citrate, Egg Yolk Tris, Skim Milk) which added vitamin C and vitamin E. The research was carried out using 4 treatments with 3 different diluent ingredients, each of which was Vitamins C and E are added to each diluent ingredient at a dose adjusted to the type of diluent agent. The treatment given is P0: Diluent without Vitamin C and E; P1: Diluent + Vitamin C; P2: Diluent + Vitamin E; and P3: Diluent + Vitamin C and Vitamin E. Research data were analyzed using analysis of variance and followed by Duncan's test at the 5% and/or 1% level for different variables. The results showed that the addition of vitamin C and vitamin E had a significant effect (P0.05) on motility and viability in the egg yolk tris diluent and egg yolk citrate but had no significant effect (P0.05) in the skim milk diluent. The use of different diluents had no significant effect (P0.05) on motility, viability, and abnormalities. This research concludes that Vitamin C added to the diluent tris egg yolk, egg yolk citrate, and skim milk showed higher average motility, percentage of live spermatozoa, and abnormalities of post-freezing Brahman cow spermatozoa.

Interplay of UV-filter pollution and temperature rise scenarios on Mytilus galloprovincialis health: Unveiling sperm quality and adult physiology, biochemistry, and histology insights

Addressing the impacts of emerging contaminants within the context of climate change is crucial for understanding ecosystem health decline. Among these, the organic UV-filters 4-methylbenzylidenecamphor (4-MBC) and benzophenone-3 (BP-3) are widely used in cosmetics and personal care products. Their unique physico-chemical properties, along with their growing commercialization and consumption, have made them ubiquitous in aquatic environments through both direct and indirect releases, raising significant concerns about their potential threats to inhabiting biota. Additionally, increasing surface water temperatures exacerbate ecological risks, making it imperative to understand the implications for non-target species at different biological levels. This study investigated the short- and long-term effects of UV-filters 4-MBC or BP-3, at ecologically relevant concentrations, combined with current and predicted warming scenarios, on the performance and male reproductive health of Mytilus galloprovincialis mussel populations. Using biomarkers across sub-cellular, cellular, tissue, and individual levels, the study revealed significant physiological and biochemical impairments in both sperm cells and adults exposed to UV-filters. Temperature emerged as the primary driver influencing mussel responses and modulating the impacts of 4-MBC/BP-3, emphasizing their sensitivity to temperatures outside the optimal range and interactive effects between stressors. Specifically, sperm motility declined with increasing UV-filter concentrations, while temperature alone influenced ROS production, leading to compromised mitochondrial activity and DNA damage in the presence of combined stressors, indicative of potential reproductive impairments. Adults exhibited high UV-filter bioconcentration potential in whole tissues, compromised physiological status, morphophysiological changes in digestive glands, oxidative stress, and alterations in metabolic capacity, antioxidant defences, and biotransformation mechanisms, correlating with UV-filter exposure and temperature increase. Among the UV-filters tested, 4-MBC was the most detrimental, especially when combined with warming. Overall, this study underscores the vulnerability of M. galloprovincialis to cumulative stressors and highlights the importance of employing a multi-biomarker approach to assess and mitigate the impacts of stressors on coastal ecosystems.