Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners’ concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have improved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent advances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular parameters. Spermatozoa protein expression already has clinical applications, for example, as a fertility and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic evaluation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation.

Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR^®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.

A novel electrode anchoring design and its implantation procedure, aiming for a minimally invasive solution for gastric electrical stimulation, are presented. The system comprises an anchor made of a flexible body embedding two needle-shaped electrodes. The electrodes can easily switch from a parallel position - to pierce the stomach - to a diverging position - enabling them to remain firmly anchored into the muscular layer of the stomach. Key device parameters governing anchoring stability were assessed on a traction test bench, and optimal values were derived. The device was then implanted in six dogs by open surgery to assess its anchoring durability in vivo. Computed tomography images showed that the electrodes remained well placed within the dogs' gastric wall over the entire assessment period (more than one year). Finally, a prototype of a surgical tool for the minimally invasive device placement was manufactured, and the anchoring procedure was tested on a dog cadaver, providing the proof of concept of the minimally invasive implantation procedure. The use of our electrode anchoring system in long-term gastric electrical stimulation is promising in terms of implantation stability (the anchor withstands a force up to 0.81 N), durability (the anchor remains onto the stomach over one year) and minimal invasiveness of the procedure (the diameter of the percutaneous access is smaller than 12 mm). Moreover, the proposed design could have clinical applications in other hollow organs, such as the urinary bladder.

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.

The increasing reliance of modern agriculture on honey bee (Apis mellifera) pollination has driven efforts to preserve and enhance bee populations. The cryopreservation of drone semen presents a promising solution for preserving genetic diversity and supporting breeding programs without live animal transport risks. This study aimed to evaluate a one-step dilution antibiotic-free drone semen slow-freezing protocol under field conditions with in vitro and in vivo parameters. Semen viability was tested by two different mixes of dyes, and both techniques gave similar results, showing a post-thaw viability drop of 37%. Virgin queens were inseminated either with fresh or frozen–thawed semen. Survival rates until egg-laying onset and female brood production were similar for both groups; however, colonies with queens inseminated with fresh semen were more likely to go into wintering. Results suggest that frozen–thawed semen can support viable queen insemination, with potential for female brood production even without antibiotics in the diluent. This study highlights the need for further refinement of cryopreservation protocols, particularly regarding semen quality and queen longevity, to improve the feasibility of cryobanking for Apis mellifera conservation and breeding.