high-throughput droplet vitrification of stallion sperm using permeating cryoprotective agents.

Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined size (i.e., ~20 µL) that were subsequently cooled by depositing on an aluminum alloy block placed in liquid nitrogen. Mathematical modeling was performed to compute the heat transfer and rate of cooling. The minimum CPA concentration needed for vitrification was determined for various CPAs (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide) and combinations thereof, while effects of droplet size and carrier solution were also identified. Sperm vitrification was eventually done using a glycerol/propylene glycol (1/1) mixture at a final concentration of 45% in buffered saline supplemented with 3% albumin and polyvinylpyrrolidon, while warming was done in standard diluent supplemented with 100 mM sucrose. The sperm concentration was found to greatly affect sperm membrane integrity after vitrification-and-warming, i.e., was found to be 21 ± 12% for 10 × 106 sperm mL-1 and 54 ± 8% for 1 × 106 sperm mL-1. However, an almost complete loss of sperm motility was observed. In conclusion, successful sperm vitrification requires establishing the narrow balance between droplet size, sperm concentration, CPA type and concentration, and exposure time.

Alginate encapsulation of stallion sperm for increasing storage stability.

The aim of this study was to establish an alginate encapsulation procedure for stallion sperm, and investigate if sperm encapsulation enhances longevity during cold storage and survival after cryopreservation. First, biocompatibility of the compounds needed for encapsulation was tested and factors determining capsule structure were identified. Sperm encapsulation was realized either by depositing droplets (20 µL) of sperm solution supplemented with barium or calcium chloride (10 mM) in alginate solution (0.25%, w/v), or by adding sperm-alginate droplets in solution containing barium or calcium chloride, and hardening (10 min). The first procedure resulted in structures with sperm residing in a liquid core surrounded by a spherical alginate shell, whereas the second procedure resulted in sperm embedded in solid beads of alginate matrix. It was found that use of calcium for alginate gelation resulted in decreased sperm motility as compared to using barium, and that encapsulation in solid beads had a negative impact on sperm plasma membrane intactness. Percentages of membrane intact sperm in barium-alginate core-shell structures were similar as found for ordinary diluted sperm, and did not change during 4 d storage at 5 °C. Sperm motility was reduced after direct recovery from core-shell structures, however, remained stable during 4 d storage leading to similar values as found for un-encapsulated sperm at this time point. Cryosurvival of sperm encapsulated in solid beads or core-shell structures was found to be lower compared to that of ordinary diluted sperm.

Towards increasing stallion sperm longevity by storage at subzero temperatures in the absence of ice.

The aim of cell preservation technologies is to slow down damaging reactions by lowering the storage temperature. Upon dilution in a stabilizing extender, stallion sperm can be stored at refrigerator temperatures for several days. Cryopreservation allows storage for decades, but freezing and thawing cause damage and viability losses. It is assumed that by storing cells at subzero temperatures in a non-frozen supercooled state, the damaging effects of ice formation can be avoided. In this study, we have investigated if stallion sperm can be stored at -10°C in the absence of ice, and compared viability during supercooled storage with that during storage at 5°C. We found that addition of 2% Ficoll-400 to buffered saline and covering with mineral oil depressed the sample freezing point and inhibited surface-catalyzed nucleation. This allowed storage in a supercooled state at -10°C for up to 7 days. Supplementing specimens with sperm, however, increased the incidence of sample freezing. Nonetheless, with 50×106 sperm mL-1, about 40% of the samples turned out to be non-frozen. Adding 100 mM sucrose was found to preserve sperm membrane intactness during supercooled storage, although this resulted in lower percentages as found with refrigerated storage. Sperm motility appeared to be lost during supercooled storage but could be partly restored by substituting buffered saline with a milk-based extender as base medium. Percentages of membrane intact sperm, however, were found to be lower. Supercooled storage holds promise for semen preservation, but further optimization of the storage solution is required to preserve sperm motility.

Occurrence of Intrauterine Purulent Concrements in a Maiden Mare-A Case Report.

Pyometra is an uncommon condition in mares associated with various symptoms. Here, we report a case of a 13-year-old Icelandic barren maiden mare with recurrent vaginal discharge. Ultrasonographically, the mare displayed intrauterine spherical masses of inhomogenous texture, which were identified as purulent concrements in hysteroscopy. The purulent concrements were successfully removed via uterine lavage after endoscope-assisted comminution. Microbiologic examination of the concrements revealed growth of Streptococcus equi subspecies zooepidemicus, Actinobacillus species, Pseudomonas aeruginosa, Staphylococcus intermedius, Pseudomonas fulva, Citrobacter freundii, and Chryseobacterium species. Systemic antibiotic treatment with trimethoprim-sulfadiazine and additional uterine lavages were performed for 10 days. A follow-up examination revealed absence of intrauterine masses but reoccurrence of pyometra due to an impatent cervical canal. The pyometra condition was resolved by insertion of a cervical stent for prevention of intrauterine fluid accumulation. In conclusion, uterine masses, which may severely impact fertility, are best diagnosed by hysteroscopy. Intrauterine purulent concrements should be considered as an atypical form of equine pyometra.