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This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C ( n  = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 ( n  = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

Abstract To increase rams’ post-thaw semen quality following cryopreservation, this study used enriched Tris-based diluent with varying amounts of moringa leaf methanolic extract (MLME). The antioxidant activity, total phenolic, and total flavonoid content were all assessed in MLME. The sperm of five healthy Awassi rams were collected, divided into 4 equal aliquots, and diluted [1:5; (v/v)] in Tris-citrate-glucose extender supplemented with 0.48, 0.56, and 0.64 mg MLME/ml or without MLME supplementation (control). The percentages of sperm total motility (STM, %), sperm progressive motility (SPM, %) and viability (V, %), abnormal morphology (AM, %), membrane functional integrity (MFI, %), and acrosome integrity (AI %) were measured. Malondialdehyde (MDA), nitric oxide (NO), ascorbic acid (AA), superoxide dismutase (SOD), glutathione peroxidase (GPx), total cholesterol (TC), low-density lipoproteins (LDL), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), zinc (Zn), and copper (Cu) were measured. The total phenolic gallic acid and flavonoid catechin (equivalent) contents were 19.78 mg/g and 11.94 mg/g, respectively. 2,2-Diphenyl-1-picrylhydrazyl (34.37 mM TE/g) and 2,2′-azino-bis/3-ethylbenzothiazoline-6-sulfonic acid (53.47 mM TE/g) were found in MLME. MLME had a 64.59 mM TE/g ferric-reducing power. In comparison to control, the addition of 0.64 mg/ml MLME to Tris-based extender resulted in the highest (P < 0.001) STM (55.22 ± 0.98), SPM (45.41 ± .70), SV (60.01 ± 1.05), MFI (75.23 ± 0.77), and AI (73.13 ± 0.72) and the lowest (P < 0.001) AM (21.34 ± 0.72) values. In comparison to the control, the addition of 0.56 mg/ml semen extender resulted in lower STM, SPM, SV, MFI, and AI with higher AM percentages. MDA (P = 0.03), NO (P = 0.012), CHO (P = 0.0001), and LDL (P = 0.004) were reduced by 0.64 mg/ml MLME, while AA (P = 0.017) and SOD (P = 0.0001) were elevated. In conclusion, the highest copper (P = 0.006) and lowest zinc concentrations in MLME (0.48 mg/ml extender) deteriorated the post-thaw semen quality, prompting us to suggest the addition of 0.64 mg MLME to rams’ Tris-based semen extender.

This study evaluated the impact of pre-freezing semen dilution rate and dimethyl acetamide (DMA) concentration on the post-thaw motility and fertility of cryopreserved rooster sperm. Rooster ejaculates were diluted with a standard EK extender to achieve low (LSC; 1 × 10⁹ sperm/mL) and high (HSC; 2 × 10⁹ sperm/mL) sperm concentrations. Each dilution group was further treated with three DMA concentrations (3%, 6%, or 9%) before cryopreservation. Post-thaw sperm motility traits were obtained by computer-assisted sperm analysis (CASA), and fertility features were evaluated through artificial insemination in hens. The current results showed that HSC significantly improved total motility (TM), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF), but reduced linearity (LIN) and straightness (STR) compared to LSC. DMA concentration had a quadratic effect on motility, with 6% yielding the highest progressive motility (PM), straight line velocity (VSL), and BCF. Fertility outcomes revealed that HSC resulted in higher fertilization rates, while neither DMA concentrations nor their interaction with dilution rates exerted significant effects on fertility traits. VCL, ALH, and BCF showed positive correlations with pipping-chicks rates, whereas STR, LIN, and WOB displayed negative correlations. These findings underscore the critical interplay between dilution rate and cryoprotectant concentration and provide practical guidance for developing more reliable cryopreservation protocols that can be applied under field conditions to enhance fertility management in poultry production.

The association between poor paternal diet, perturbed embryonic development, and adult offspring ill health represents a new focus for the Developmental Origins of Health and Disease hypothesis. However, our understanding of the underlying mechanisms remains ill-defined. We have developed a mouse paternal low-protein diet (LPD) model to determine its impact on semen quality, maternal uterine physiology, and adult offspring health. We observed that sperm from LPD-fed male mice displayed global hypomethylation associated with reduced testicular expression of DNA methylation and folate-cycle regulators compared with normal protein diet (NPD) fed males. Furthermore, females mated with LPD males display blunted preimplantation uterine immunological, cell signaling, and vascular remodeling responses compared to controls. These data indicate paternal diet impacts on offspring health through both sperm genomic (epigenetic) and seminal plasma (maternal uterine environment) mechanisms. Extending our model, we defined sperm- and seminal plasma-specific effects on offspring health by combining artificial insemination with vasectomized male mating of dietary-manipulated males. All offspring derived from LPD sperm and/or seminal plasma became heavier with increased adiposity, glucose intolerance, perturbed hepatic gene expression symptomatic of nonalcoholic fatty liver disease, and altered gut bacterial profiles. These data provide insight into programming mechanisms linking poor paternal diet with semen quality and offspring health.

Sperm quality is often evaluated through computer-assisted semen analysis (CASA) and is an indicator of boar fertility. The aim of this research was to study the relationship between CASA motility parameters and fertility results in pigs. Insemination records and semen parameters from a total of 45,532 ejaculates collected over a 3-yr period were used. The statistical model for analysis of fertility data from these inseminations included factors related to sow productivity. The boar- and semen-related variance (direct boar effect) were corrected for the effects of individual boar, genetic line of the boar, age of the boar, days between ejaculations, number of sperm cells in an ejaculate, number of sperm cells in an insemination dose, and AI station. The remaining variance was analyzed if semen motility parameters had a significant effect. This analysis revealed significant (P < 0.05) effects of progressive motility, velocity curvilinear, and beat cross frequency on farrowing rate (FR). Total motility, velocity average path, velocity straight line, and amplitude of lateral head displacement affected (P < 0.05) total number of piglets born (TNB). Boar- and semen-related parameters explained 5.3% of the variation in FR and 5.9% of the variation in TNB. Motility parameters, measured by CASA, explained 9% of the boar- and semen-related variation in FR and 10% of the boar- and semen-related variation in TNB. Individual boar and genetic line of the boar affected (P < 0.0001) the variation in FR and TNB. No differences (P > 0.05) were observed between effects of AI stations on fertility outcome, underscoring the objectivity of the CASA system used. Motility parameters can be measured with CASA to assess sperm motility in an objective manner. On the basis of the motility pattern, CASA enables one to discriminate between the fertilizing capacity of ejaculates, although this depends on the genetic line of the boar used in AI stations.

Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC–MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences. Summary sentence Acquisition of freezability of boar sperm occurs during holding time and involves changes in energy metabolism pathways. Graphical Abstract Creatine can be phosphorylated by ATP in Creatine phosphate, mainly at the mitochondria. Creatine phosphate is an alternative source for ADP phosphorylation in ATP. The Hypoxanthine-Inosine pathway is a different source of AMP–ADP–ATP conversion. Spermine increases the activation of cAMP, which can increase the energy production by the electron transport chain. Niacinamide is a precursor of NAD+, playing a role in the Krebs cycle and the electron transport chain. The 2-Methylbutyrylcarnitine is a product of isoleucine degradation and a source of succinate. GEF 0 h—good ejaculate freezer cryopreserved after 0 h of holding time at 17°C; GEF 24 h—good ejaculate freezer cryopreserved after 24 h of holding time at 17°C; PEF 0 h—poor ejaculate freezer cryopreserved after 0 h of holding time at 17°C; PEF 24 h— poor ejaculate freezer cryopreserved after 24 h of holding time at 17°C.

Bluetongue virus serotype 3 (BTV-3) emerged in northwestern Europe in 2023–2024, raising concerns about its potential reproductive impact on rams, similar to previous outbreaks with BTV-8. This study assessed the effect of natural BTV-3 infection on the semen quality of 49 rams in Belgium using two cross-sectional sampling sessions during the 2024 outbreak. Semen and blood were tested for BTV RNA via RT-qPCR, and a composite semen quality score (SQS) was established based on key sperm parameters. On the first sampling date, 75% of rams were viremic, and 19% presented azoospermia. Rams with BTV RNA detectable in both semen and blood had significantly lower SQS and sperm concentrations than those with viral RNA in blood only or none at all. By the second sampling, 53 days later, semen quality had improved markedly, indicating a transient effect of infection. These findings confirm that BTV-3 can severely but temporarily impair ram fertility, particularly when viral replication occurs in the reproductive tract. Given the seasonal overlap between vector activity and breeding programs, these results underscore the importance of integrating reproductive health monitoring into outbreak response strategies.

Vaccination-induced allergic reactions in bulls raise both body and testicular temperatures, leading to germ cell damage, epididymal dysfunction, accelerated testicular ageing, and increased sperm abnormalities that ultimately degrade semen quality. This study aimed to ameliorate vaccination stress and improve semen quality using meloxicam and levamisole. The present study was conducted at the Artificial Breeding Research Centre, ICAR-NDRI, Karnal, Haryana, with twelve Sahiwal breeding bulls that were divided into four groups: Group I (Control), Group II (Meloxicam), Group III (Levamisole), and Group IV (Meloxicam + Levamisole). Semen parameters were evaluated during pre- and post-vaccination. Treated groups showed significant improvement ( p  < 0.05) in initial progressive motility, live sperm, HOST, and acrosome integrity compared to the control, with the highest improvement observed in the combination group (meloxicam + levamisole). Sperm abnormalities, moribund, dead, apoptotic sperm, and lipid peroxidation were significantly higher in the control group. The meloxicam + levamisole group exhibited significantly lower ( p  < 0.05) sperm abnormalities and lipid peroxidation. Improvement was observed in semen quality with treatment (Meloxicam + levamisole-treated bulls) in percentage of live (+ 53.11%), moribund (− 33.83%), dead (− 36.70%), apoptotic spermatozoa (− 44.01%), and DNA fragmentation index (− 47.07%) in relation to the control groups on the 7th post vaccination day.