Cooling dynamics of droplets exposed to solid surface freezing and vitrification.

Solid surface freezing or vitrification (SSF/SSV) can be done by depositing droplets of a sample, e.g., cells in a preservation solution, onto a pre-cooled metal surface. It is used to achieve higher cooling rates and concomitant higher cryosurvival rates compared to immersion of samples into liquid nitrogen. In this study, numerical simulations of SSF/SSV were conducted by modeling the cooling dynamics of droplets of cryoprotective agent (CPA) solutions. It was assumed that deposited droplets attain a cylindrical bottom part and half-ellipsoidal shaped upper part. Material properties for heat transfer simulations including density, heat capacity and thermal conductivity were obtained from the literature and extrapolated using polynomial fitting. The impact of CPA type, i.e., glycerol (GLY) and dimethyl sulfoxide (DMSO), CPA concentration, and droplet size on the cooling dynamics was simulated at different CPA mass fractions at temperatures ranging from -196 to 25 °C. Simulations show that glycerol solutions cool faster compared to DMSO solutions, and cooling rates increase with decreasing CPA concentration. However, we note that material property data for GLY and DMSO solutions were obtained in different temperature and concentration ranges under different conditions, which complicated making an accurate comparison. Experimental studies show that samples that freeze have a delayed cooling response early on, whereas equilibration times are similar compared to samples that vitrify. Finally, as proof of concept, droplets of human red blood cells (RBCs) were cryopreserved using SSV/SSF comparing the effect of GLY and DMSO on cryopreservation outcome. At 20% (w/w), similar hemolysis rates were found for GLY and DMSO, whereas at 40%, GLY outperformed DMSO.

Preserving frozen stallion sperm on dry ice using polymers that modulate ice crystalization kinetics.

Cryopreserved semen is routinely shipped in liquid nitrogen. Dry ice could serve as an alternative coolant, however, frozen storage above liquid nitrogen temperatures (LN2, -196 °C) may negatively affect shelf-life and cryosurvival. In this study, we determined critical temperatures for storage of cryopreserved stallion sperm. We evaluated: (i) effects of cooling samples to different subzero temperatures (-10 °C to -80 °C) prior to storing in LN2, (ii) stability at different storage temperatures (i.e., in LN2, dry ice, -80 °C and -20 °C freezers, 5 °C refrigerator), and (iii) sperm cryosurvival during storage on dry ice (i.e., when kept below -70 °C and during warming). Furthermore, (iv) we analyzed if addition of synthetic polymers (PVP-40, Ficoll-70) modulates ice crystallization kinetics and improves stability of cryopreserved specimens. Sperm motility and membrane intactness were taken as measures of cryosurvival, and an artificial insemination trial was performed to confirm fertilizing capacity. We found that adding PVP-40 or Ficoll-70 to formulations containing glycerol reduced ice crystal sizes and growth during annealing. Post-thaw sperm viability data indicated that samples need to be cooled below -40 °C before they can be safely plunged and stored in LN2. No negative effects of relocating specimens from dry ice to LN2 and vice versa became apparent. However, sample warming above -50 °C during transport in dry ice should be avoided to ensure preservation of viability and fertility. Moreover, addition of PVP-40 or Ficoll-70 was found to increase sperm cryosurvival, especially under non-ideal storage conditions where ice recrystallization may occur.

Flow cytometric detection and identification of different leukocyte subpopulations in stallion semen.

To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups, 27.85 ± 5.7% of events were positive for CD4, CD8, CD21 or CD172a, while in native semen samples, leukocytes were scarce (0.114 ± 0.134%). The most abundant leukocyte subpopulation in semen was of lymphoid origin (CD4-positive cells [0.015 ± 0.02%]), whereas CD21-positive cells (B cells; 0.001 ± 0.001%) were virtually absent in ejaculates of fertile stallions. This presented flow cytometric assay for the detection and identification of different leukocyte population in equine antioxidant-treated ejaculates can be used as an additional tool for spermatological examination in stallions.

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.

Factors Affecting the Membrane Permeability Barrier Function of Cells during Preservation Technologies.

Cellular membranes are exposed to extreme conditions during the processing steps involved in cryopreservation (and freeze-drying) of cells. The first processing step involves adding protective agents. Exposing cells to protective agents causes fluxes of both water and solutes (i.e., permeating cryoprotective agents) across the cellular membrane, resulting in cell volume changes and possibly osmotic stress. In addition, protective molecules may interact with lipids, which may lead to membrane structural changes and permeabilization. After loading with protective agents, subsequent freezing exposes cells to severe osmotic and mechanical stresses, caused by extra and/or intracellular ice formation and a drastically increased solute concentration in the unfrozen fraction. Furthermore, cellular membranes undergo thermotropic and lyotropic phase transitions during cooling and freezing, which drastically alter the membrane permeability and its barrier function. In this article, it is shown that membrane permeability to water and solutes is dependent on the temperature, medium osmolality, types of solutes present, cell hydration level, and absence or presence of ice. Freezing most drastically alters the membrane permeability barrier function, which is reflected as a change in the activation energy for water transport. In addition, membranes become temporarily leaky during freezing-induced fluid-to-gel membrane phase transitions, resulting in the uptake of impermeable solutes.

Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions.

Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6M concentrations compared to only 1M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.

Freezing-induced uptake of trehalose into mammalian cells facilitates cryopreservation.

The aim of this study was to investigate if membrane-impermeable molecules are taken up by fibroblasts when exposing the cells to membrane phase transitions and/or freezing-induced osmotic forces. The membrane-impermeable fluorescent dye lucifer yellow (LY) was used to visualize and quantify uptake during endocytosis, and after freezing-thawing. In addition, trehalose uptake after freezing and thawing was studied. Fourier transform infrared spectroscopic studies showed that fibroblasts display a minor non-cooperative phase transition during cooling at suprazero temperatures, whereas cells display strong highly cooperative fluid-to-gel membrane phase transitions during freezing, both in the absence and presence of protectants. Cells do not show uptake of LY upon passing the suprazero membrane phase transition at 30-10°C, whereas after freezing and thawing cells show intracellular LY equally distributed within the cell. Both, LY and trehalose are taken up by fibroblasts after freezing and thawing with loading efficiencies approaching 50%. When using 250 mM extracellular trehalose during cryopreservation, intracellular concentrations greater than 100 mM were determined after thawing. A plot of cryosurvival versus the cooling rate showed a narrow inverted-'U'-shaped curve with an optimal cooling rate of 40°C min(-1). Diluting cells cryopreserved with trehalose in isotonic cell culture medium resulted in a loss of cell viability, which was attributed to intracellular trehalose causing an osmotic imbalance. Taken together, mammalian cells can be loaded with membrane-impermeable compounds, including the protective agent trehalose, by subjecting the cells to freezing-induced osmotic stress.

Freezing-induced uptake of disaccharides for preservation of chromatin in freeze-dried stallion sperm during accelerated aging.

Nonviable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a glassy state. Their use is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sperm when exposed to freezing. Trehalose uptake likely occurs during freezing-induced membrane phase transitions. Stallion sperm was freeze-dried in various formulations consisting of reducing or nonreducing sugars combined with albumin as bulking agent. Chromatin stability was studied during storage at 37°C, using the flow cytometric sperm chromatin structure assay and microscopic assessment of chromatin dispersion and DNA fragmentation after electrophoresis. Freeze-drying did not affect sperm chromatin, irrespective of the formulation that was used. DNA fragmentation index (DFI) values ranged from 5 to 8%. If sperm was freeze-dried without protectants or in a combination of glucose and proteins, DNA damage rapidly accumulated during storage at 37°C, reaching DFI values of respectively 95 ± 4 and 64 ± 42% after 1 month. DFI values of sperm freeze-dried with sucrose or trehalose ranged between 9-11% and 33-52% after 1 and 3 months storage, respectively. In conclusion, freeze-drying sperm with disaccharides results in uptake during freezing, which greatly reduces chromatin degradation during dried storage.

Induced sub-lethal oxidative damage affects osmotic tolerance and cryosurvival of spermatozoa.

If the physiological balance between production and scavenging of reactive oxygen species (ROS) is shifted towards production of ROS this may result in accumulation of cell damage over time. In this study stallion spermatozoa were incubated with xanthine and xanthine oxidase (X-XO) to artificially generate defined levels of superoxide and hydrogen peroxide resulting in sub-lethal oxidative damage. The effects of X-XO treatment on various sperm characteristics were studied. Special emphasis was placed on sperm osmotic tolerance pre-freeze and its correlation with cryosurvival, given that cryopreservation exposes cells to osmotic stress. ROS accumulation occurred predominantly in the sperm midpiece region, where the mitochondria are located. Exposing spermatozoa to increasing X-XO concentrations resulted in a dose-dependent decrease in sperm motility. Percentages of plasma membrane-intact spermatozoa were not affected, whereas stability of membranes towards hypotonic stress decreased with increasing levels of induced oxidative stress. Infrared spectroscopic studies showed that X-XO treatment does not alter sperm membrane phase behaviour. Spermatozoa exposed to higher oxidative stress levels pre-freeze exhibited reduced cryosurvival. Centrifugation processing and addition of catalase were found to have little beneficial effect. Taken together, these results show that treatment of spermatozoa with X-XO resulted in different levels of intracellular ROS, which decreased sperm osmotic tolerance and cryosurvival.

Screening of whole genome sequences identified high-impact variants for stallion fertility.

BACKGROUND: Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions. METHODS: Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). RESULTS: We screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant g.37455302G>A in NOTCH1 with the de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). For 9 high-impact variants within the genes CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absence of the homozygous mutant genotype in the validation sample of all 337 fertile stallions was obvious. Therefore, these variants were considered as potentially deleterious factors for stallion fertility. CONCLUSIONS: In conclusion, this study revealed 17 genetic variants with a predicted high damaging effect on protein structure and missing homozygous mutant genotype. The g.37455302G>A NOTCH1 variant was identified as a significant stallion fertility locus in Hanoverian stallions and further 9 candidate fertility loci with missing homozygous mutant genotypes were validated in a panel including 19 horse breeds. To our knowledge this is the first study in horses using next generation sequencing data to uncover strong candidate factors for stallion fertility.

Intrafollicular Oocyte Transfer (IFOT) of Abattoir-Derived and In Vitro-Matured Oocytes Results in Viable Blastocysts and Birth of Healthy Calves.

There are still major differences between in vitro production (IVP)-derived and in vivo-derived bovine blastocysts. Therefore, intrafollicular oocyte transfer (IFOT) was used in the present study to allow early embryonic development within the physiological oviductal environment, in order to avoid subsequent harmful effects of the in vitro culture environment. Using modified ovum pickup equipment, in vitro-matured oocytes were transferred into the preovulatory follicle of synchronized heifers (follicular recipients), enabling subsequent ovulation, in vivo fertilization, and in vivo development. When 1646 in vitro-matured oocytes were transferred to 28 follicular recipients, a total of 583 embryos (35.2%) were recovered in excess after uterine flushing at Day 7. Although numbers of generated extra embryos were highly variable, preovulatory follicles with a diameter of 13-14 mm delivered significantly (P < 0.05) larger amounts of extra embryos (34.3 vs. 7.3), as well as extra morulae and blastocysts (8.3 vs. 0.8), compared with follicles with a diameter of 9-10 mm. Nevertheless, the developmental rate to the blastocyst stage was lower in IFOT compared with in vitro-derived control (Vitro) embryos at Day 7 (8.0% vs. 36.5%). Likewise, cumulative developmental rates to the morula or blastocyst stage until Day 7 were lower in IFOT-derived embryos when related to the number of transferred (8.4% vs. 51.7%) or flushed (22.8% vs. 51.7%) embryos. Of the latter, IFOT-derived embryos yielded significantly lower cleavage rates compared with the Vitro controls (63.2% vs. 88.8%), and developmental rate to the morula or blastocyst stage were lower even when related to the proportion of cleaved embryos (36.8% vs. 58.2%). In contrast, lipid content and cryotolerance did not differ between IFOT and fully IVP embryos; but IFOT-derived embryos showed significantly lower lipid content (P < 0.05) and significantly higher cryotolerance compared with IVP-derived embryos cultured in CR1aa medium supplemented with estrus cow serum (ECS), but not when cultured in SOFaa medium supplemented with fatty acid-free BSA (BSA-FFA). Finally, transfer of 19 frozen-thawed IFOT-derived blastocysts to synchronized recipients (uterine recipients) resulted in pregnancy rates comparable with those obtained after transfer of fully in vivo-derived embryos or IVP-derived embryos cultured in SOFaa + BSA-FFA, whereas pregnancy rate following transfer of IVP-derived blastocysts was significantly lower when they were cultured in CR1aa + ECS (42.1% vs. 13.8%). All in all, seven pregnancies presumed to be IFOT derived went to term, and microsatellite analysis confirmed that five calves were indeed derived from IFOT. To our knowledge, these are the first calves born after IFOT in cattle. Interestingly, the average birth weight of IFOT-derived calves was lower than that of IVP-derived calves, even when embryos were cultured in SOFaa + BSA-FFA, indicating that the environment during early embryo development might cause fetal overgrowth. Taken together, for the first time we were able to show that IFOT is a feasible technique to generate bovine blastocysts by transferring in vitro-matured oocytes derived from slaughterhouse ovaries. These IFOT-derived blastocysts closely resemble in vivo-derived blastocysts in terms of lipid content and freeze survival. Thus, the present study laid the groundwork for newly created scientific experiments enabling novel analytical possibilities. Nevertheless, IFOT-derived embryos still reached lower pregnancy rates by trend compared with in vivo-derived embryos, also implicating an important role for the maturational environment in further developmental characteristics.

Tolerance of spermatozoa to hypotonic stress: role of membrane fluidity and correlation with cryosurvival.

The aim of this study was to evaluate inter-individual variability in osmotic properties of stallion spermatozoa and its correlation with cryosurvival. In addition, temperature dependency of hypo-osmotic tolerance and membrane fluidity were studied. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15-30 °C temperature range, whereas membrane stability was found to be decreased at 4 and 37 °C. Bull spermatozoa showed greater hypo-osmotic tolerance compared with stallion spermatozoa, especially at temperatures above 30 °C, which coincided with decreased membrane fluidity of bovine spermatozoa in this temperature range. The critical osmolality at 22 °C, at which half of the sperm population survived exposure to hypotonic saline solution, was found to vary between 55 and 170 mOsm kg(-1) among different stallions. Clear correlations were found for pre- versus post-freeze sperm motility and membrane integrity. Pre-freeze percentages of membrane-intact spermatozoa after exposure to hypotonic stress showed a weak correlation with sperm motility after cryopreservation. This correlation, however, was not found when data were corrected for initial numbers of membrane-intact spermatozoa in the sample. We thus conclude that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions.

Developmental competence of equine oocytes: impacts of zona pellucida birefringence and maternally derived transcript expression.

In the present study, equine oocytes were classified into groups of presumably high and low developmental competence according to cumulus morphology, as well as oocyte ability to metabolise brilliant cresyl blue (BCB) stain. All oocytes were evaluated individually in terms of morphometry, zona pellucida birefringence (ZPB) and relative abundance of selected candidate genes. Oocytes with an expanded cumulus (Ex), representing those with presumably high developmental competence, had a significantly thicker zona (18.2 vs 17.3µm) and a significantly higher ZPB (64.6 vs 62.1) than oocytes with a compacted cumulus (Cp). Concurrently, oocytes classified as highly developmentally competent (BCB+) had a significantly thicker zona (18.8 vs 16.1µm) and significantly higher ZPB (63.1 vs 61.3) compared with oocytes classified as having low developmental competence. Expression of TFAM, STAT3 and CKS2 was significantly higher in Ex compared with Cp oocytes, whereas expression of COX1, ATPV6E and DNMT1 was lower. Together, the data reveal that developmentally competent equine oocytes are larger in size, have higher ZPB values and exhibit a typical genetic signature of maternally derived transcripts compared with oocytes with lower in vitro developmental competence.

Success of different therapies for bacterial endometritis in stud farm practice.

Bacterial endometritis is a major problem in equine reproduction usually treated with antibiotics, however reports of success rates are scarce. This study collected data from mares diagnosed with intrauterine bacterial growth and compared the outcome of different therapies for bacterial endometritis in German stud farm practice. Data on mares with positive uterine culture results were collected retrospectively in veterinary practices (n = 5; 2018-2022). Information relating to 30 factors (mare, diagnostics, therapy, pregnancy rate) of bacterial endometritis cases (n = 772) were recorded and analyzed. Possible effects on treatment success (positive pregnancy result in the first cycle after treatment) were tested by binomial logistic regression. In most cases ß-hemolytic streptococci were detected (n = 707). Treatments for the endometritis included trimethoprim-sulfonamides (n = 409), procaine-penicillin (n = 227), marbofloxacin (n = 53) or no antibiotics (n = 59) and most antibiotics were administered systemically (n = 711) rather than locally (n = 23). Uterine lavage was reported in 49 % of mares. Uterotonic drugs were administered in 42.2 % of mares. Breeding programs included artificial insemination (AI) with chilled semen (n = 667), AI with frozen semen (n = 169), transfer of fresh (n = 112) or cryopreserved (n = 27) embryos and natural cover (n = 27). In the first cycle after treatment, the pregnancy rate was 47 % and it rose to 69 % by end of the season. Treatment success was affected by duration of antibiotic treatment, veterinary practice, and presence of clinical signs. In conclusion, reported treatment practices in German stud farm practice resulted in acceptable pregnancy results and the multiple binomial logistic regression approach identified factors affecting the pregnancy outcome in this dataset.

Use of membrane transport models to design cryopreservation procedures for oocytes.

Oocyte cryopreservation is increasingly being used in reproductive technologies for conservation and breeding purposes. Further development of oocyte cryopreservation techniques requires interdisciplinary insights in the underlying principles of cryopreservation. This review aims to serve this purpose by: (1) highlighting that preservation strategies can be rationally designed, (2) presenting mechanistic insights in volume and osmotic stress responses associated with CPA loading strategies and cooling, and (3) giving a comprehensive listing of oocyte specific biophysical membrane characteristics and commonly used permeation model equations. It is shown how transport models can be used to simulate the behavior of oocytes during cryopreservation processing steps, i.e., during loading of cryoprotective agents (CPAs), cooling with freezing as well as vitrification, warming and CPA unloading. More specifically, using defined cellular and membrane characteristics, the responses of oocytes during CPA (un)loading were simulated in terms of temperature- and CPA type-and-concentration-dependent changes in cell volume and intracellular solute concentration. In addition, in order to determine the optimal cooling rate for slow programmable cooling cryopreservation, the freezing-induced cell volume response was simulated at various cooling rates to estimate rates with tolerable limits. For vitrification, special emphasis was on prediction of the timing of reaching osmotic tolerance limits during CPA exposure, and the need to use step-wise CPA addition/removal protocols. In conclusion, we present simulations and schematic illustrations that explain the timing of events during slow cooling cryopreservation as well as vitrification, important for rationally designing protocols taking into account how different CPA types, concentrations and temperatures affect the oocyte.

Frequency of potentially pathogenic bacterial and fungal isolates among 28,887 endometrial samples from mares, with an emphasis on multi-drug resistant bacteria in Germany (2018-2022).

Antimicrobial-resistant bacteria pose a serious threat to the wellbeing of animals and humans. In equine reproduction, endometritis caused by facultative microbial pathogens is a condition, which is usually treated with antibiotics. Data from Germany on prevalence of facultative pathogenic microorganisms cultured in samples from the equine uterus and the frequency of multi-drug resistant (MDR) bacteria is lacking. The aim of the study was to provide representative numbers for both. Microbiological culture results (n = 28,887) of endometrial samples submitted to a large veterinary diagnostic laboratory from 2018-2022 were analyzed. An average of 25.9 % of the culture results showed growth of facultative pathogenic bacteria. The dominant isolated bacteria were ß-hemolytic streptococci (79.7 %) followed by Escherichia (E.) coli variatio haemolytica (5.2 %). E. coli were cultured in 4.3 % of the samples and occurred more often than Klebsiella pneumoniae (3.9 %), Candida species (2.9 %), Pseudomonas aeruginosa (2.0 %), and Staphylococcus aureus (1.5 %). Antibiotic susceptibility testing revealed sensitivity of ß-hemolytic streptococci towards penicillins in almost 100 % of the cultured samples (99.5 %). E. coli-isolates were sensitive to gentamicin in 96.2 % of the cases. The frequency of multidrug-resistant extended spectrum beta-lactamase (ESBL)-positive bacteria and methicillin-resistant Staphylococcus aureus (MRSA) was 3.1 % of all positive culture results. The number of ESBL-positive isolates (n = 159) and MRSA was stable from 2018-2022. In conclusion, the situation regarding occurrence of MDR bacteria in Germany is favorable, but should further be monitored.

Success rates and factors influencing pregnancy outcome after 464 transvaginal ultrasound-guided twin reductions in the mare.

BACKGROUND: Transvaginal ultrasound-guided aspiration (TUA) is used for post-fixation twin reduction in mares. However, there is limited information regarding factors that influence pregnancy outcome after TUA. OBJECTIVES: To evaluate the effect of day of gestation on which TUA is performed, aspiration volume, puncture of the conceptus, medication administered before and after TUA, embryo location, mare age and parity and operator experience on pregnancy and foaling rates after TUA. STUDY DESIGN: Retrospective case series. METHODS: Data were collected from case records of 464 TUAs performed by 14 operators in 422 mares diagnosed pregnant with dizygotic twins in two different facilities between 2010 and 2019. Pregnancy status was determined by ultrasonography at 5-7 days and 3-4 weeks after the TUA was performed. Subsequent pregnancy and foaling results were obtained by follow-up communication. The effects of mare, gestation- and TUA-related variables on pregnancy and foaling rates were analysed by the chi-square-test for homogeneity and Fisher's exact test and logistic regression. RESULTS: TUA was performed between 21 and 82 days of gestation in unilaterally (267/359 [74.4%]) and bilaterally fixed (92/359 [25.6%]) twin pregnancies. A singleton pregnancy (218/381 [57.2%]), persistent twin pregnancy (60/381 [15.8%]), or the loss of both conceptuses (103/381 [27%]) was confirmed 5-7 days after TUA was performed. At 3-4 weeks post TUA 50.3% (163/324) of mares were diagnosed with a single viable pregnancy and 40.1% (127/317) went on to deliver a live single foal. TUA performed early in gestation (D 25-35) resulted in the birth of a live singleton foal in 49.3% (74/150) of mares. MAIN LIMITATIONS: Missing retrospective data despite extensive follow-up. CONCLUSION: This is the first large scale study to demonstrate that acceptable pregnancy and foaling rates can be achieved in mares diagnosed with twins when TUA is performed early in gestation (<40 days).

Analysis of gene and protein expression in the endometrium for validation of an ex vivo model of the equine uterus using PCR, digital and visual histopathology.

In the past, most research in equine reproduction has been performed in vivo but the use of in vitro and ex vivo models has recently increased. This study aimed to evaluate the functional stability of an ex vivo hemoperfused model for equine uteri with molecular characterization of marker genes and their proteins. In addition, the study validated the respective protein expression and the aptness of the software QuPath for identifying and scoring immunohistochemically stained equine endometrium. After collection, uteri (n = 12) were flushed with preservation solution, transported to the laboratory on ice, and perfused with autologous blood for 6 h. Cycle stage was determined by examination of the ovaries for presence of Graafian follicles or corpora lutea and analysis of plasma progesterone concentration (estrus: n = 4; diestrus: n = 4; anestrus: n = 4). Samples were obtained directly after slaughter, after transportation, and during perfusion (240, 300, 360 min). mRNA expression levels of progesterone (PGR), estrogen (ESR1) and oxytocin (OXTR) receptor as well as of MKI67 (marker of cell growth) and CASP3 (marker of apoptosis) were analyzed by RT-qPCR, and correlation to protein abundance was validated by immunohistochemical staining. Endometrial samples were analyzed by visual and computer-assisted evaluation of stained antigens via QuPath. For PGR, effects of the perfusion and cycle stage on expression were found (P < 0.05), while ESR1 was affected only by cycle stage (P < 0.05) and OXTR was unaffected by perfusion and cycle stage. MKI67 was lower after 360 min of perfusion as compared to samples collected before perfusion (P < 0.05). For CASP3, differences in gene expression were found after transport and samples taken after 240 min (P < 0.05). Immunohistochemical staining revealed effects of perfusion on stromal and glandular cells for steroid hormone receptors, but not for Ki-67 and active Caspase 3. OXTR was visualized in all layers of the endometrium and was unaffected by perfusion. Comparison of QuPath and visual analysis resulted in similar results. For most cell types and stained antigens, the correlation coefficient was r > 0.5. In conclusion, the isolated hemoperfused model of the equine uterus was successfully validated at the molecular level, demonstrating stability of key marker gene expression. The utility of computer-assisted immunohistochemical analysis of equine endometrial samples was also confirmed.

Osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents.

The aim of this study was to determine how different membrane-permeable and -impermeable cryoprotective agents modulate tolerance of stallion sperm to osmotic stress and stabilize membranes during cryopreservation. Special emphasis was on hydroxyl ethylene starch (HES), which exposes cells to minimal osmotic stress due to its large molecular weight. Percentages of motile sperm post-thaw were found to be similar when glycerol, sucrose, and HES were used at their optimal concentrations. Percentages of plasma membrane intact sperm after return to isotonic medium were highest for HES. Fourier transform infrared spectroscopy studies were carried out to study subzero membrane phase and permeability behavior. Cryoprotectants were shown to decrease the initial rate of membrane dehydration during freezing, decrease the activation energy for water transport, and increase the total extent of freezing-induced dehydration. Freezing studies with liposomes as a model system showed that only the membrane-permeable cryoprotective agents glycerol and ethylene glycol protected membranes against leakage, whereas egg yolk, sucrose, and HES did not. Differential scanning calorimetry studies showed that sucrose and HES raise the glass transition temperature of the freezing extender and the difference in heat capacity associated with the glass transition. This indicates that these compounds enable formation of a stable glassy matrix at higher subzero temperatures. Sperm cryosurvival rates can be increased by combining different cryoprotectants with different protective functions; membrane permeable cryoprotective agents stabilize membranes and modulate the rate of cellular dehydration, whereas di- and polysaccharides increase the glass transition temperature and facilitate storage and handling at higher subzero temperatures.

Freezing-induced cellular and membrane dehydration in the presence of cryoprotective agents.

FTIR and cryomicroscopy have been used to study mouse embryonic fibroblast cells (3T3) during freezing in the absence and presence of DMSO and glycerol. The results show that cell volume changes as observed by cryomicroscopy typically end at temperatures above -15°C, whereas membrane phase changes may continue until temperatures as low as -30°C. This implies that cellular dehydration precedes dehydration of the bound water surrounding the phospholipid head groups. Both DMSO and glycerol increase the membrane hydraulic permeability at subzero temperature and reduce the activation energy for water transport. Cryoprotective agents facilitate dehydration to continue at low subzero temperatures thereby decreasing the incidence of intracellular ice formation. The increased subzero membrane hydraulic permeability likely plays an important role in the cryoprotective action of DMSO and glycerol. In the presence of DMSO water permeability was found to be greater compared to that in the presence of glycerol. Two temperature regimes were identified in an Arrhenius plot of the membrane hydraulic permeability. The activation energy for water transport at temperature ranging from 0 to -10°C was found to be greater than that below -10°C. The non-linear Arrhenius behavior of Lp has been implemented in the water transport model to simulate cell volume changes during freezing. At a cooling rate of 1°C min(-1), ∼5% of the initial osmotically active water volume is trapped inside the cells at -30°C.