Assessment Of Sperm Quality Research Articles

Association Between Zinc Ion Concentrations in Seminal Plasma and Sperm Quality: A Chinese Cross-Sectional Study.

This study investigates the impacts of zinc ion concentration in in seminal plasma and the total amount of Zn2+ per ejaculation on sperm quality evaluation parameters. In addition, we assessed the reliability of using zinc content in seminal plasma to evaluate sperm quality. We analyzed semen from 964 men and found that men over 40years old had significantly lower concentrations of Zn2+ in ejaculated semen compared to other age groups (p < 0.05), with no significant difference in Zn2+ concentrations among other age groups (p > 0.05). The total amount of Zn2+ in one ejaculation did not show a statistical difference between the normal semen and the abnormal semen groups (p > 0.05). Statistical differences were observed in sperm kinetic parameters and DNA fragmentation index between the normal Zn2+ total amount group and the abnormal group (p < 0.05). However, there was no statistical difference in morphological parameters (p > 0.05). Spearman correlation analysis showed a negative correlation between Zn2+ concentrations in seminal plasma, age, and sperm fructose and a positive correlation between semen volume, abstinence time, sperm concentration, neutral α-glycosidase, and citric acid content (p < 0.05). The receiver operating characteristic curve analysis demonstrated that Zn2+ concentrations had poor accuracy and specificity in assessing sperm quality (p > 0.05). Although there is a partial correlation between Zn2+ concentrations in seminal plasma and certain semen quality parameters, relying solely on Zn2+ concentrations to evaluate sperm quality lacks accuracy and specificity.

Effects of Resveratrol-Loaded Cyclodextrin on the Quality Characteristics of Ram Spermatozoa Following Cryopreservation.

This study investigated the effects of pure and methyl-β-cyclodextrin loaded forms of resveratrol (10 µg/mL, 20 µg/mL, and 40 µg/mL) on ram sperm functions post-thawing. Semen samples were pooled and divided into ten groups: Control, RES10, RES20, RES40, CD10, CD20, CD40, RLC10, RLC20, and RLC40. The groups were pre-diluted with media containing the group-specific chemicals, followed by 15 min of incubation, dilution, and freezing. To assess the effects of the chemicals, a post-thaw sperm quality assessment was conducted. Motility and other velocity parameters were evaluated using computer-assisted semen analysis. The functional integrity of spermatozoa membranes was assessed with the hypo-osmotic swelling test, and the capacitation status of spermatozoa was determined through fluorescent microscopic evaluation. Additionally, flow cytometry was used to evaluate mitochondrial activity, oxidative stress, and the integrity of the sperm membrane and acrosome. The results indicated that cyclodextrin adversely affected sperm functions following freezing-thawing, notably increasing the rate of spermatozoa exhibiting pre-capacitation and mitochondrial activity by approximately 34% and 16%, respectively (p < 0.05). It was found that 20 µg/mL resveratrol prevented pre-capacitation (p < 0.05). Both resveratrol and resveratrol-loaded cyclodextrin groups improved post-thaw sperm qualities overall, demonstrating their utility for freezing ram semen. However, higher concentrations of resveratrol were found to negatively impact sperm functions.

Androgenic effects of the aqueous extract of Pycnanthus angolensis (Welw.) Warb. (Myristicaceae) wood in hemicastrated male Wistar rats

Background: Androgen deficiency is the most common disorder of reproductive function and can lead to male sexual disorders. Objective: This study was designed to evaluate the androgenic effects of Pycnanthus angolensis (Welw.) Warb in hemicastrated rats. Materials and methods: Forty-two male rats were divided into 6 groups of 7 rats each, including a group of uncastrated rats that received distilled water (10 ml/kg); a group of castrated rats that received 10 ml/kg of distilled water; a group of castrated rats that received testosterone enanthate (3 mg/kg BW) per week intramuscularly; and 3 groups of castrated rats that received 43, 86 and 172 mg/kg of the aqueous extract of Pycnanthus angolensis, respectively. After 14 days of oral treatment, the rats were killed by decapitation. The blood was collected and the androgen-dependent organs were collected for histological sectioning, and biochemical analysis. The tail of the epididymis was used to assess sperm quality. Results: Treatment with the aqueous extract at doses of 43 and 86 mg/kg, significantly improved the sexual behavior of castrated rats, with increases of 25.92% and 22.74% intromissions frequency, and 67.06% and 56.46% mount frequency, compared to those in the castrated rats which did not receive any treatment. The extract also enhanced sperm quality in castrated rats. Both doses also significantly increased serum testosterone levels with rates of 45.07% and 49.00%, respectively; compared to those in the negative control group. Conclusion: In view of the aforesaid results, Pycnanthus angolensis (Welw.) Warb could be considered as a promising natural agent in hypogonadism management. Keywords: Androgen, hypogonadism, castrated male rats, unilateral castration, Pycnanthus angolensis (Welw.) Warb

O-308 Is AI the future of sperm quality assessment? A comparative study of AI-enhanced and conventional semen analysis systems

Abstract Study question How does the accuracy of an artificial intelligence (AI)-enhanced semen analysis system compare to conventional semi-automated methods when assessing semen quality? Summary answer Compared to conventional semi-automated methods for sperm quality assessment, the AI-enhanced system tends to overestimate progressive sperm motility and normal sperm morphology. What is known already Male factor infertility, specifically poor sperm quality, accounts for over 40% of all infertility cases. Accurate semen analysis is thus a critical step in guiding appropriate treatment strategies in male reproductive health. Past decades have witnessed the development of semi-automated systems for semen analysis, overcoming the limitations of traditional microscopy. While these systems offer precise data on kinetics and concentration, manual intervention remains necessary for assessing certain parameters, potentially affecting reproducibility. AI-based systems have been recently introduced into clinical practice, promising more objective and standardized analysis. Yet, the extent to which these methods surpass conventional approaches remains to be established. Study design, size, duration This is a single-centre, prospective, consecutive, paired study, conducted from February to September 2022. The study included 84 sperm samples from patients undergoing medically assisted reproduction. Participants/materials, setting, methods All sperm samples were analyzed using the AI-enhanced automated system (LENSHooke X1 Pro, Bonraybio®, Fertil Ibérica) and conventional methods: CASA semi-automatized device (ISAS, Proiser), Diff-Quik staining for manual morphology assessment, and pH test strips. We measured and compared several semen parameters, including morphology (% normal sperm), concentration (million/mL), motility (% progressive, % non-progressive, % total motility), and pH, using both approaches. Spearman and Kruskal-Wallis tests were used for statistical analyses. Main results and the role of chance We observed significant differences among several semen parameters when comparing the AI-enhanced system and conventional approaches. Mean normal sperm morphology, as assessed by the AI-enhanced automated device, was significantly higher than that evaluated by conventional microscopy (4.62% versus 3.07%, p &amp;lt; 0.00001). We also observed significant differences for motility and pH parameters (57.98 versus 25.66, p = 0.0001 and 7.86 versus 8.38, p = 0.0001, respectively). Correlations between variables assessed using both methods also varied. Sperm concentration showed a strong correlation between the AI-enhanced and conventional systems (Spearmann’s Rho=0.828), however a weak correlation was observed for total and progressive motility, as well as the percent of immotile sperm (69.54% versus 28.98% R2=0.541; 57.98% versus 25.66% R2=0.566, and 30.45% versus 47.12%, R2=0.541, respectively). There was no significant correlation for non-progressive motility between the two methods (6.53% versus 11.60%, R2=0.157). Limitations, reasons for caution This study exclusively compared two analysis methods among numerous AI-enhanced systems available on the market. Given the rapidly evolving landscape of AI technologies, caution is warranted when generalizing these findings to other AI models, as their performance may vary. Wider implications of the findings Observed differences between the AI-enhanced system and conventional methods require careful consideration. While fully automated semen analysis systems are designed to reduce subjectivity, extensive comparative research is crucial. A comprehensive market assessment will be essential for safeguarding patient treatment outcomes and advancing AI capabilities in semen analysis. Trial registration number not applicable

P-032 Prospective and multi-site analysis of pregnancy outcomes in 488 patients who completed the Sperm Quality Test (SpermQT) measuring the utility of sperm epigenetics

Abstract Study question Do SpermQT results prospectively correlate with fertility outcomes of couples undergoing intrauterine insemination (IUI)? Summary answer We observe a decrease in IUI pregnancy rates in men with Abnormal compared to Normal SpermQT results irrespective of their sperm total motile count (TMC). What is known already As the mainstay of male infertility diagnosis, the standard semen analysis provides limited value for predicting pregnancy outcomes. New technologies are needed for a more comprehensive assessment of male fertility. A novel, epigenetic assessment of sperm quality, SpermQT, was implemented clinically in 2022. Retrospective studies have shown that sperm health, as identified through DNA methylation patterns, is predictive of IUI outcomes in a subset of infertile men. Additional prospective research is needed for validation of these data. Study design, size, duration Over an 18-month period (June 2022 - January 2024), 23 reproductive endocrinologists over eight clinics ordered 488 SpermQT tests on male patients. For these patients, physicians and clinical support staff were asked to provide the sperm total motile count (TMC) from the semen analysis, male and female ages, procedure information and outcomes. SpermQT results were categorized as Normal or Abnormal. Associations between SpermQT results, outcomes, age, and TMC were analyzed. Participants/materials, setting, methods The selection of patients for SpermQT assessment was based on the clinical decision of the ordering physician. SpermQT requires a semen sample which was collected by the patient either at the andrology laboratory or at home using an at-home collection kit. All sample types were shipped directly to the vendor’s laboratory for analysis and report generation. Following the completion of the epigenetic analysis, all SpermQT results were provided to the ordering physician for patient consultation. Main results and the role of chance 488 patients received a SpermQT result. 83.6% of men had a Normal SpermQT (n = 408), and 16.4% had an Abnormal SpermQT (n = 80). 180 couples had pregnancy outcomes from at least one IUI. Of the 180, 14% of men had an Abnormal SpermQT (n = 25) and 86% had a Normal SpermQT (n = 155). Analysis of pregnancy outcomes from all 180 couples who completed one, two, or three IUI cycles showed a 15% IUI cumulative pregnancy rate. The men with a Normal SpermQT had a 17.42% cumulative IUI pregnancy rate within the first three IUI cycles while men with an Abnormal SpermQT (N = 25) had no reported pregnancies and a 0% cumulative IUI pregnancy rate. Within this patient cohort, 338 patients had reported TMC where 82% had a TMC ≥20 million (n = 276). 72% of men with an Abnormal SpermQT had a TMC ≥20M, while 84% of men with a Normal SpermQT had a TMC ≥20M. Median male and female ages in the Normal SpermQT category were both 34, while median male and female ages in the Abnormal SpermQT category were 32 and 33 respectively, indicating age did not play a role in IUI pregnancy outcomes. Limitations, reasons for caution Limitations within this study include the sample size of men with an Abnormal SpermQT result. In order to identify a statistical significance between IUI pregnancy outcomes, a total sample size of 500 patients will be needed. Data and outcomes will continue to be collected to continue the validation of SpermQT. Wider implications of the findings There is strong data showing that sperm epigenetics play an important role in IUI outcomes. Here we show in a prospective and multi-site population that a new assessment of sperm epigenetics (SpermQT) identifies men less likely to achieve an IUI pregnancy, and also identifies subfertile men with normal TMC. Trial registration number Non Applicable

P-023 Gene expression in human sperm as biomarkers and tools for the assessment of male infertility

Abstract Study question Can gene expression as AURKA, HDAC4, and CARHSP1 be used for the diagnosis of sperm quality exploring male infertility? Summary answer The comprehensive outcome of gene expression involving AURKA, HDAC4, and CARHSP1 serves as a key to classifying sperm and predicting their functionality. What is known already Diagnostic male infertility is largely based on semen parameters according to the World Health Organization (WHO) reference values. While these parameters assess sperm quality, they do not provide insights into sperm quality and have a limited predictive value on natural fecundity, fertilization rates, and assisted reproductive technology (ART) outcomes. Study design, size, duration This prospective study investigates the gene expression involved in mitosis, epigenetic regulation, and early embryo development. From February to June 2023, we included all men aged from 20 to 60 years having sperm parameters analysis in our unit center. A total of 277 semen samples were collected, 129 (46.4%) exhibiting three normal sperm parameters according to WHO criteria. Among these, 32 samples showed oligo-astheno-teratozoospermia (OAT), while 116 samples had 1 or 2 abnormal parameters. Participants/materials, setting, methods The mRNA expression levels of three candidate genes (AURKA, HDAC4, and CARHSP1) were quantified in fresh ejaculate semen using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The thresholds for the spermatozoa function index (SFI) were established by assessing the combined expression of these target genes, considering the morphology and the capability to reach a blastocyst stage. The sperm were ranked according to the concentration (million/ml), motility (%) and morphology (%). Main results and the role of chance We assess the quality of spermatozoa for three biomarkers by analyzing their mRNA expression levels. Receiver operator characteristics curves (ROC) indicate a combined index value expression. This model establishes an index threshold &amp;gt; 320 for an “over-index value,” distinguishing spermatozoa with a normal capacity, an index threshold &amp;lt; 290 for an “under-index value,” and 290 &amp;lt; index &amp;lt; 320 for an “intermediate-index value”. Among the 277, we find 112 (40.4%) have an over-expression index; 161 (58.1%) have an under-expressed index and 4 (1.4%) have an intermediate-index. Interestingly, among the 129 samples with the three normal WHO parameters, we show that 82 (63.5%) have an over-index value, 44 (34.1%) have an under-index value, and 3 (2.3%) display an intermediate-index value. In addition, all the 32 OAT samples have an under-index. These findings suggest that the OAT sperm population implies a potential epigenetic network connected with our three target genes. Furthermore, even in cases with normal sperm parameters, there may be an under-expressed index, indicating lower functionality in terms of fertilization and early embryo development. Limitations, reasons for caution The present method was validated only on ejaculate semen in men free of any chronic disease and any anti-oxidant treatment. This prospective study requires further validation in the largest population. Wider implications of the findings This study’s results could highlight understanding a chaotic early embryo development in ART and used to explore male infertility; particularly in unexplained infertility. Trial registration number not applicable

Evaluation of the morphological and functional state of cryopreserved sperm of bulls at different periods of long-term storage

Objective. To study the morphological andfunctional state of cryopreserved sperm of bulls during storage for 6 - 27 years at a temperature of - 196 C in liquid nitrogen in a comparative aspect. Methods. The object of the study was cryopreserved semen of bulls stored for 6 - 10 or 20 - 27 years. In the study of the qualitative parameters offrozen- thawed spermatozoa, the method of visual assessment for motility and survival, the method ofers differential staining with trypan blue/Gymza dyes to determine the morphological and functional integrity of cell membranes and the test for the ability to bind to vesicular explants of bovine oviductal epithelial cells in vitro were used. Results. It was found that the extension of the storage period of spermatozoa to 20 - 27years had no significant effect on the functional characteristics o f spermatozoa in terms of motility, survival and absolute survival. The method of differential staining showed that the proportion of live sperm with an intact acrosome in the total distribution did not differ significantly in both studied storage periods and ranged from 61.6 - 63.9 %. The proportion of dead cells also did not differ significantly and was 26.4 % when stored for up to 10 years and 21.7 % when stored for more than 20 years. No significant difference was found in the overall assessment of the percentage of sperm with varying degrees of acrosomal membrane damage. This figure was 22.5 and 26.0 %, respectively. At the same time, when the shelf life of cryopreserved sperm was extended to 20 - 27 years, a significant increase in the percentage of live sperm with a missing acrosome was observed (p&lt;0.01). It was determined that the index of sperm binding to explants of epithelial cells of the oviduct in vitro was significantly higher when storedfor 6-10 years and was 227.9±32.3 against 114.6±19.5 when stored for 20 - 27 years (p&lt;0.05). Conclusions. The use of the traditional method of assessing sperm quality by indicators of sperm motility, survival and absolute sperm survival did not reveal any differences when storing cryopreserved sperm for 6 - 27 years. At the same time, the differential staining method showed a significant increase of 7.2% in the proportion of live sperm with a missing acrosome when the storage period was extended from 6 - 10 to 20 - 27 years. The in vitro test for sperm binding to oviduct epithelial cells showed a significant decrease of almost 2 times in the index of sperm binding to cellular explants during long-term storage. This may indicate a decrease in the ability of such sperm to fertilise. Keywords: bulls, sperm, cryopreservation, long-term storage, differential colouration, oviduct, sperm binding.

CP-Net: Instance-aware part segmentation network for biological cell parsing

Instance segmentation of biological cells is important in medical image analysis for identifying and segmenting individual cells, and quantitative measurement of subcellular structures requires further cell-level subcellular part segmentation. Subcellular structure measurements are critical for cell phenotyping and quality analysis. For these purposes, instance-aware part segmentation network is first introduced to distinguish individual cells and segment subcellular structures for each detected cell. This approach is demonstrated on human sperm cells since the World Health Organization has established quantitative standards for sperm quality assessment. Specifically, a novel Cell Parsing Net (CP-Net) is proposed for accurate instance-level cell parsing. An attention-based feature fusion module is designed to alleviate contour misalignments for cells with an irregular shape by using instance masks as spatial cues instead of as strict constraints to differentiate various instances. A coarse-to-fine segmentation module is developed to effectively segment tiny subcellular structures within a cell through hierarchical segmentation from whole to part instead of directly segmenting each cell part. Moreover, a sperm parsing dataset is built including 320 annotated sperm images with five semantic subcellular part labels. Extensive experiments on the collected dataset demonstrate that the proposed CP-Net outperforms state-of-the-art instance-aware part segmentation networks.

Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in 'Land of Fires', Italy.

Can illegal discharge of toxic waste into the environment induce a new condition of morpho-epigenetic pathozoospermia in normozoospermic young men? Toxic environmental contaminants promote the onset of a new pathozoospermic condition in young normozoospermic men, consisting of morpho-functional defects and a sperm increase of low-quality circular RNA (circRNA) cargo, tightly linked to contaminant bioaccumulation in seminal plasma. Epidemiological findings have reported several reproductive anomalies depending on exposure to contaminants discharged into the environment, such as germ cell apoptosis, steroidogenesis defects, oxidative stress induction, blood-testis barrier dysfunctions, and poor sperm quality onset. In this scenario, a vast geographical area located in Campania, Italy, called the 'Land of Fires', has been associated with an excessive illegal discharge of toxic waste into the environment, negatively impacting human health, including male reproductive functions. Semen samples were obtained from healthy normozoospermic men divided into two experimental groups, consisting of men living in the 'Land of Fires' (LF; n = 80) or not (CTRL; n = 80), with age ranging from 25 to 40 years. The study was carried out following World Health Organization guidelines. Quality parameters of semen from CTRL- and LF-normozoospermic men were evaluated by computer-assisted semen analysis; high-quality spermatozoa from CTRL and LF groups (n = 80 for each experimental group) were obtained using a 80-40% discontinuous centrifugation gradient. Seminal plasma was collected following centrifugation and used for the dosage of chemical elements, dioxins and steroid hormones by liquid chromatography with tandem mass spectrometry. Sperm morpho-functional investigations (cellular morphology, acrosome maturation, IZUMO1 fertility marker analysis, plasma membrane lipid state, oxidative stress) were assessed on the purified high-quality spermatozoa fraction by immunochemistry/immunofluorescence and western blot analyses. Sperm circRNA cargo was evaluated by quantitative RT-PCR, and the physical interaction among circRNAs and fused in sarcoma (FUS) protein was detected using an RNA-binding protein immunoprecipitation assay. Protein immunoprecipitation experiments were carried out to demonstrate FUS/p-300 protein interaction in sperm cells. Lastly, in vitro lead (Pb) treatment of high-quality spermatozoa collected from normozoospermic controls was used to investigate a correlation between Pb accumulation and onset of the morpho-epigenetic pathozoospermic phenotype. Several morphological defects were identified in LF-spermatozoa, including: a significant increase (P < 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P < 0.01) of peanut agglutinin and IZUMO1 null signal cells. In agreement with these data, abnormal steroid hormone levels in LF seminal plasma suggest a premature acrosome reaction onset in LF-spermatozoa. The abnormal immunofluorescence signals of plasma membrane cholesterol complexes/lipid rafts organization (Filipin III and Flotillin-1) and of oxidative stress markers [3-nitrotyrosine and 3-nitrotyrosine and 4-hydroxy-2-nonenal] observed in LF-spermatozoa and associated with a sperm motility reduction (P < 0.01), demonstrated an affected membrane fluidity, potentially impacting sperm motility. Bioaccumulation of heavy metals and dioxins occurring in LF seminal plasma and a direct correlation between Pb and deregulated circRNAs related to high- and low-sperm quality was also revealed. In molecular terms, we demonstrated that Pb bioaccumulation promoted FUS hyperacetylation via physical interaction with p-300 and, in turn, its shuttling from sperm head to tail, significantly enhancing (P < 0.01 versus CTRL) the endogenous backsplicing of sperm low-quality circRNAs in LF-spermatozoa. Participants were interviewed to better understand their area of origin, their eating habits as well as their lifestyles, however any information incorrectly communicated or voluntarily omitted that could potentially compromise experimental group determination cannot be excluded. A possible association between seminal Pb content and other heavy metals in modulating sperm quality should be explored further. Future investigations will be performed in order to identify potential synergistic or anti-synergistic effects of heavy metals on male reproduction. Our study provides new findings regarding the effects of environmental contaminants on male reproduction, highlighting how a sperm phenotype classified as normozoospermic may potentially not match with a healthy morpho-functional and epigenetic one. Overall, our results improve the knowledge to allow a proper assessment of sperm quality through circRNAs as biomarkers to select spermatozoa with high morpho-epigenetic quality to use for ART. This study was supported by 'Convenzione Azienda Sanitaria Locale (ASL) Caserta, Regione Campania' (ASL CE Prot. N. 1217885/DIR. GE). The authors have no conflict of interest to declare. N/A.

Effect of Three Semen Extenders on Sperm Quality and In Vitro Fertilization Rates of Fresh and Cryopreserved Sperm Collected from Llama (Lama glama) Vas Deferens.

The advances in Assisted Reproductive Technologies (ARTs) applied in South American camelid species are still scarce. The aim of this study was to compare the effects of three semen extenders, before and after the cryopreservation of spermatozoa obtained from the vas deferens, on sperm quality parameters and in vitro fertilization rates of llama (Lama glama) oocytes. Mature fertile llama males (Lama glama; n = 6; age: 48-60 mo.; BCS: ~2.7) were included in the study. Sperm samples were collected from each male using the surgical technique of the vas deferens deviation. Then, the sperm samples were pooled and diluted with the Tris-EY, Andromed®, or BioxCell® extender in order to subsequently carry out the sperm cryopreservation process. The sperm quality assessment related to each extender was performed before and after cryopreservation with regard to sperm morphological abnormalities, acrosome integrity, sperm viability, membrane permeability, and sperm motility traits. Moreover, in vitro fertilization (IVF) procedures were carried out to evaluate the in vitro fertility of the cryopreserved sperm samples using each extender. Overall, significant differences were observed before and after cryopreservation regarding acrosome integrity, sperm viability, membrane permeability, and sperm motility traits among the extenders used, where Tris-EY and Andromed® were better than BioxCell® (p < 0.05); however, no differences were observed regarding the sperm morphological abnormalities among extenders (p > 0.05). Moreover, multiple differences were observed with regard to the velocity and linearity kinematic parameters obtained by computerized analysis before and after the cryopreservation process, irrespective of the extender used (p < 0.05). Finally, differences were observed regarding the in vitro fertilization rates among the different extender-derived samples (p < 0.05). In conclusion, the sperm quality using Tris-EY and Andromed® was better before and after cryopreservation compared to that using BioxCell®. Although the number of fertilized oocytes obtained after the IVF process between Tris-EY and Andromed® was similar, Andromed®-derived samples showed the best sperm quality results before and after cryopreservation. This indicates that the cryopreservation extender is a determining factor in significantly improving in vitro fertilization rates when using sperm samples obtained from vas deferens in llama (Lama glama) males.

An Overview of Artificial Insemination: A Journey from Past to Present

Artificial insemination (AI) has a rich history spanning centuries, beginning with early experiments in plant and animal reproduction. This article traces the development of AI from its earliest recorded instances in 1322 A.D. to modern-day advancements. Notable pioneers such as Spallanzani, Hunter, and Ivanoff contributed significantly to the evolution of AI techniques. The introduction of AI across various animal species, including cattle, horses, sheep, goats, pigs, and poultry, revolutionized breeding practices worldwide. Key milestones include the development of semen extenders, the invention of artificial vaginas for semen collection, and the advent of cryopreservation techniques. Moreover, advancements in sperm quality assessment and estrous synchronization further enhanced AI efficiency. The introduction of sexed semen technology has provided a means to control offspring gender, offering new avenues for genetic selection. Overall, AI has played a crucial role in improving livestock genetics, disease control, and reproductive efficiency, making significant contributions to agricultural productivity and sustainability.

Post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions.

Artificial insemination with cooled-shipped semen is the primary method used in the equine breeding industry; yet, sperm quality and fertility can be suboptimal for some stallions when standard techniques are used. Therefore, there is a critical need to develop alternative approaches for these stallions. To assess sperm quality parameters and fertility of cooled-stored stallion semen processed by SpermFilter® or centrifugation and resuspended in three extenders. Controlled and field study. In Experiment 1, semen was collected from 21 stallions classified as having good ('Good-coolers', n = 8) or poor ('Bad-coolers', n = 13) semen cooling. The semen was extended at 30 million spermatozoa/mL in a skimmed milk-based (SM) diluent, and refrigerated for 24 h. Then, the cooled-stored semen was processed through SpermFilter® or centrifugation, and the resulting sperm pellets were resuspended in SM, SM containing pentoxifylline (SM-P), or an egg yolk-based (EY) extender. Unprocessed cooled-stored semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. Experiment 2, cooled semen from 9 stallions classified as Bad-coolers was used to inseminate 18 embryo donor mares at 66 cycles (Unprocessed, n = 22; SpermFilter®/SM-P, n = 16; or SpermFilter®/EY, n = 28). Data were analysed with a mixed model and Tukey's as posthoc, and logistic regression. Processed semen resuspended in EY had superior sperm motility compared to unprocessed, SM and SM-P (p < 0.0001). Semen processed by SpermFilter® resuspended in SM-P was similar to EY (p > 0.05). Pellet resuspension with EY and SM-P improved the HMMP of Bad-cooler stallions (p = 0.0010). Semen processed by SpermFilter® had superior PMI to centrifuged semen (p < 0.0001). Mares inseminated with SpermFilter®/SM-P (50%, 8/16) or SpermFilter®/-EY (68%, 9/28) had higher pregnancy rates than mares bred with unprocessed semen (14%, 3/22) (p < 0.001). Low number of mares in the fertility trial. Sperm quality and fertility of Bad-cooler stallions can be enhanced by SpermFilter® and pellet resuspension with either EY or SM-P.

The Adaptation Time to the Extender as a Crucial Step for an Accurate Evaluation of Ram Sperm Quality during the Liquid Storage.

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.

Evaluation of the Congjiang Xiang Pig’s Sperm Quality by Flow Cytometry

Background: Sperm quality plays an important role in the animal industry. The study of the sperm quality of Congjiang Xiang pigs is limited and the Rh123/PI double staining has not been used to detect the sperm quality of boars. Thus, the study aimed to evaluate the sperm quality of the Congjiang Xiang pigs and the feasibility of the flow cytometry (FCM) method in assessing sperm quality in pigs. Methods: Semen samples were evaluated for routine indicators of semen quality and using FCM with single staining and double staining to analyze the integrity of membrane and acrosome, the mitochondrial function. The sperm quality of the Congjiang Xiang pigs was compared with the Large White pigs. Result: The semen of the Congjiang Xiang pigs had a lower sperm concentration than that of the Large White pigs (P less than 0.01). There were no significant differences in sperm vitality, sperm motility and abnormality between the two breeds (P greater than 0.05). Single sperm staining revealed a lower sperm acrosomal integrity in the semen of the Congjiang Xiang pigs than that of the Large White pigs (P less than 0.01). No significant difference between the two breeds in membrane integrity and mitochondrial function was observed (P greater than 0.05). Rh123/PI double staining demonstrated that the rate of live sperm with a normal mitochondrial function of the Congjiang Xiang pigs was significantly higher than that of the Large White pigs (P less than 0.05). The rate of necrotic sperms in the semen of the Congjiang Xiang pigs was significantly lower than that in the semen of the Large White pigs (P less than 0.05). Correlation analysis revealed a positive association between the rates of live sperm in the semen of the congjiang xiang pigs and their sperm motility (P=0.051). Our results suggest that Rh123/PI double staining with flow cytometry can provide more reliable information to assess the boar sperm quality.

The future of equine semen analysis.

We are currently experiencing a period of rapid advancement in various areas of science and technology. The integration of high throughput 'omics' techniques with advanced biostatistics, and the help of artificial intelligence, is significantly impacting our understanding of sperm biology. These advances will have an appreciable impact on the practice of reproductive medicine in horses. This article provides a brief overview of recent advances in the field of spermatology and how they are changing assessment of sperm quality. This article is written from the authors' perspective, using the stallion as a model. We aim to portray a brief overview of the changes occurring in the assessment of sperm motility and kinematics, advances in flow cytometry, implementation of 'omics' technologies, and the use of artificial intelligence/self-learning in data analysis. We also briefly discuss how some of the advances can be readily available to the practitioner, through the implementation of 'on-farm' devices and telemedicine.

(203) Impact of Smoking on Sperm Chromatin Integrity

Abstract Introduction Semen analysis is an essential component of infertility evaluation, but it may still fail to detect subtle sperm defects present in patients with male factor infertility. Although estimates vary, approximately 15% of patients with male factor infertility have normal spermiograms It is necessary to identify diagnostic measures for male infertility that are easy to perform, inexpensive, and provide an accurate diagnosis. Tests that assess sperm quality should not only identify the ability of spermatozoa to reach the oocyte but also their ability to fertilize the oocyte and activate embryo growth. The estimation of sperm DNA damage fills the above need in the diagnosis of male infertility Abnormalities in the male genome characterized by damaged sperm DNA may be indicative for male subfertility regardless of routine semen parameters, and these parameters do not reveal sperm DNA defects Men with normal semen analysis may also have a high degree of DNA fragmentation, which can be a major cause of undiagnosed/unexplained infertility. Sperm DNA fragmentation may result from aberrant chromatin packaging during spermatogenesis, defective apoptosis before ejaculation or excessive production of reactive oxygen species (ROS) in the ejaculate. Objective To study the impact of cigarette smoking on sperm chromatin integrity in infertile patients. Methods The present study included eighty infertile male patients recruited from the Andrology Outpatient Clinic at Al-Azhar University Hospitals The patients were divided into two groups: - Group I: forty nonsmoker infertile males. (Control Group). Group II: forty cigarette smoker infertile males. (Study Group) Results The current study revealed that smoking increases the abnormality of sperm chromatin integrity in sperms of smokers. This is evident by the positive correlation of increased head stainability of sperms in smokers compared to nonsmokers. The value of partially stained sperm heads using aniline blue is calculated from the total staining score formula, this score was found to increase significantly among smokers in comparison to nonsmokers. In the present study, the increased severity of smoking (smoking index), was not correlated with the degree of sperm head stainability. This mean that, smoking insult on sperm chromatin integrity is crucial regardless the duration or the amount of the consumed cigarettes Conclusions There is a significant increase of sperm chromatin damage in smoker infertile patients than nonsmoker infertile patients and these damages may affect the quality of the ejaculated spermatozoa and decrease their fertility potential. Not only semen parameters but also sperm chromatin condensation is affected significantly by smoking. Aniline blue staining is an effective and inexpensive tool for assessment of the fertilization potentiality of infertile patients. A significant correlation was found between the percentage of spermatozoa with normal sperm chromatin condensation and normal morphology. Cigarette smoking may have a negative impact on sperm quality and DNA integrity. Therefore, it would be advisable for men to abstain from smoking to avoid decreased fertility. Disclosure No.

Exploring the full potential of sperm function with nanotechnology tools.

Sperm quality is essential to guarantee the success of assisted reproduction. However, selecting high-quality sperm and maintaining it during (cryo)preservation for high efficiency remains challenging in livestock reproduction. A comprehensive understanding of sperm biology allows for better assessment of sperm quality, which could replace conventional sperm analyses used today to predict fertility with low accuracy. Omics approaches have revealed numerous biomarkers associated with various sperm phenotypic traits such as quality, survival during storage, freezability, and fertility. At the same time, nanotechnology is emerging as a new biotechnology with high potential for use in preparing sperm intended to improve reproduction in livestock. The unique physicochemical properties of nanoparticles make them exciting tools for targeting (e.g., sperm damage and sexing) and non-targeting bioapplications. Recent advances in sperm biology have led to the discovery of numerous biomarkers, making it possible to target specific subpopulations of spermatozoa within the ejaculate. In this review, we explore potential biomarkers associated with sperm phenotypes and highlight the benefits of combining these biomarkers with nanoparticles to further improve sperm preparation and technology.

Adverse Effects of Hydroxychloroquine on Testicular Tissue and Sperm Quality: An Experimental study on Male Albino Rats

Introduction: Hydroxychloroquine (HCQ) is a medicine that has mainly been used to treat malaria. During the recent Coronavirus Disease-2019 (COVID-19) period, an abrupt spike in the sale of HCQ has been found. The repurposing of this drug in the treatment of COVID-19 was not only found to be ineffective but also showed many side effects. The adverse effects of the enormous exposure to this drug should be unveiled. Aim: To evaluate the acute effect of HCQ exposure on the male reproductive system by analysing the changes in antioxidant enzyme levels in testicular tissue, sperm quality, and histopathological assessment of testicular tissue using male albino rats as the subject model. Materials and Methods: The present experimental study was done by using male albino rats as the subject model. It was conducted in the laboratory of the Department of Human Physiology at Raja NL Khan Women’s College (Autonomous) in Medinipur, West Bengal, Kolkata, India, from the last week of June 2023 to the first week of August 2023. A total 10 male albino rats were obtained and randomly divided into two groups (n=5 in each group), control and HCQ-treated, and were given plain drinking water and 33 mg/kg/day HCQ, respectively, for six consecutive days through oral gavage. After the treatment tenures, rats were euthanised, and antioxidant enzymatic activity was measured by a spectrophotometer. Sperm quality and testicular histopathology were studied. A two-tailed t-test was used to compare the parameters of the control and HCQ-treated groups. Results: The total testicular weight of the control group was 2.37±0.17 g, in contrast treated group had a significantly lower testicular weight of 1.98±0.19 g. A significant decrease in total testicular protein in the treated group was found, i.e., 28.86±3.03 mg/g of tissue, compared to the control group, i.e., 60.46±2.85 mg/g of tissue. The sperm count of the control group was 4.51±0.06 million/ml of suspension, while the treated group had a significantly lower sperm count, i.e., 2.73±0.11 million/mL of suspension. The percentages of sperm motility and viability in the control group were 86±1.63% and 94±1.33%, respectively, but in the treated group, the percentages were significantly lower, i.e., 22.66±1.35% and 24±1.78%, respectively. The increasing sperm anomalies after HCQ exposure indicate poor sperm quality. Conclusion: Infertility or reduced fertility can result from testicular dysfunction, in continuation, decrease in sperm count, motility, viability, together with abnormalities in sperm morphology, was observed after this exposure. Thus, the present study provides insight into the adverse effects of the use of this drug, which might be associated with male infertility.

Specific setup and methodology for computer-assisted sperm analysis (CASA) in evaluating elasmobranch sperm

1.One-third of elasmobranch species are threatened with extinction, and the precise assessment of their sperm parameters and quality is one stage in the development of reproductive technology for animal conservation.2.Computer-Assisted Sperm Analysis (CASA) is the gold standard for sperm analysis; however, there were not one setup established for elasmobranchs.3.This study presents the description of a specific setup developed for elasmobranch semen analysis using the CASA system.4.In addition, it provides a gold-standard technique protocol for elasmobranch sperm quality assessment.5.The described setup presented an accuracy of more than 90 percent in fourteen distinct species of freshwater and marine sharks, stingrays, and rays, failing only when there were two or more bundled sperm.

The characterization of CellROX™ probes could be a crucial factor in ram sperm quality assessment.

Several authors have demonstrated that low levels of reactive oxygen species (ROS) are necessary for the physiological functions of sperm, such as capacitation, hyperactivation, acrosomal reaction and fertilization. However, high levels of ROS are associated with oxidative stress and detrimental effects on fertility. Consequently, deep characterization of ROS presence using different fluorescent probes could be crucial. In this sense, the study of intracellular ROS localization and the relationships between ROS and other conventional parameters could improve the characterization of sperm quality for semen preservation protocols in rams. In this work, a multiparametric study was carried out by analyzing four experimental groups of ram sperm with different initial qualities: fresh semen (from both breeding and nonbreeding seasons), frozen-thawed semen and, a positive control group treated with hydrogen peroxide (300 μM) as a marker of extreme damage. Sperm analyses, including viability, apoptosis, lipid peroxidation, motility and kinetic parameters, were applied to compare several experimental groups with different sperm qualities. After that, the signals from two different ROS probes: CellROX™ Deep Red (CRDR) and Green (CRG), were examined by flow cytometry (percentage of cells that express ROS) and fluorescence microscopy (intracellular ROS location). Comparing conventional parameters, fresh samples from the breeding season showed the highest sperm quality, while the positive control samples showed the worst sperm quality. Concerning the ROS probes, the CRDR levels were higher in fresh samples from the breeding season than in the positive control and cryopreserved samples. Surprisingly, CRG presented its highest level (P < 0.05) in the positive control group treated with peroxide by flow cytometry. CRDR and CRG presented opposite labeling patterns that were corroborated by fluorescence microscopy, which determined that the probes localized in different parts of sperm. CRDR was found in the sperm mitochondrial region, while CRG was observed in the cell nucleus, suggesting that ROS localization is an important factor. Finally, our study indicates that CRDR is correlated with proper viability and sperm motility, and could be associated with high mitochondrial activity, while CRG is associated with sperm damage.