Search

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. [ABSTRACT FROM AUTHOR]

The use of cryopreserved semen has become a standard technique in equine reproduction and is significantly affected by the stallion’s individual aptitude for the technique. The aim of the study was to compare the effect of different semen handling protocols before cryopreservation on post-thaw semen quality of stallions (n=9) either classified as good (n=5) or poor freezers (n=4). Ejaculates (n=6) were collected, splitted and subjected to five different protocols: Group 1 - ejaculates frozen immediately after collection and centrifugation at room temperature (25°C); Group 2 - ejaculates frozen after cooling at 5°C for 6 hours with centrifugation before storage; Group 3 - ejaculates frozen after cooling at 5°C for 6 hours (slow cooling: 0.1°C/min) without centrifugation before storage; Group 4 - ejaculates frozen after cooling at 5°C for 24 hours with centrifugation before storage ; Group 5 - ejaculates frozen after cooling at 5°C for 24 hours (slow cooling: 0.1°C/min) without centrifugation before storage. Spermatological examination (directly before freezing (T1) and after thawing (T2) included assessment of motility, membrane integrity, acrosomal status and DNA fragmentation index (DFI). Differences between good and bad freezers were observed in all groups. While good freezers had the highest post-thaw progressive motility (T2PM) in Group 1 and 2, poor freezers showed the best results for T2PM in Group 3. Cooled storage for 24 hours resulted in lower T2PM for all groups. Post-thaw DFI and membrane integrity were not affected by treatment group but freezability of the individual stallion. More acrosomal defects were observed in poor freezers as compared to good freezers and after cooled storage for 24h in poor freezers in comparison to cooled storage for 6 hours. In conclusion, good freezers showed the best post-thaw semen quality after immediate centrifugation and cryopreservation or after centrifugation, followed by cooled storage for 6 hours. Post-thaw semen quality of poor freezers benefitted from slow cooling and cooled storage (6 hours) without previous centrifugation. [ABSTRACT FROM AUTHOR]

The objectives of the present study were to analyze systematic effects on semen quality traits from fresh and frozen-thawed semen collected in and outside season. A total of 4,681 reports on semen traits of 121 stallions representing Arabian, Thoroughbred, Quarter Horse and four warmblood breeds used for artificial insemination at the Lower Saxon National stud Celle were edited for analysis of gel-free volume, sperm concentration, total number of sperm and total number of motile sperm in fresh semen and in frozen-thawed semen progressive motility, DNA fragmentation index and non-viable sperm. Month, year, age, breed and stallions effects were analyzed with a linear mixed model procedure. Breed differences were significant for sperm concentration, total number of sperm, total motile sperm number and DNA fragmentation index. Hanoverian stallions showed significant higher least squares means for sperm concentration, total number of sperm and total motile sperm number in fresh semen, whereas Thoroughbred had significant higher least squares means for DNA fragmentation index. Stallions with an age of 2–6 years had significant lower least squares means in sperm concentration, total number of sperm and total motile sperm number and progressively motile sperm post-thawing than the other age groups. Month was significant for all semen traits but progressively motile sperm post-thawing. Month by age class interaction showed a significant influence. Inter-stallion variance accounted for 27–71% and the two-way stallion by month interaction for 2–7% of the total variance of semen quality traits. The largest proportion of inter-individual variance among stallions was obtained for the DNA fragmentation index. • Thorougbred stallions showed highest values for DNA fragmentation index among seven horse breeds. • Seasonal changes were significant for all semen traits with exception of progressive motile sperm post-thawing. • The stallion variance accounted for 27–71% of the total variance of post-thaw semen traits. • Stallion by month variance was small with estimates at 2–7% for fresh and post-thaw semen traits. [ABSTRACT FROM AUTHOR]

Semen analysis is a valuable approach to assess male fertility in the horse. The aim of this study was to evaluate the effects of two different semen collection protocols, stallion's age, season and previous rearing conditions on sperm parameters of young stallions. Semen samples were obtained from 23 stallions (age range: 30-42 months old) every six weeks. Stallions were reared under different conditions prior to the start of the study. Semen collection was either performed twice with an interval of one hour on a given day (protocol A) or once daily for seven days (protocol B). The ejaculates were examined for total sperm number (TSN), progressive motility (PM), plasma membrane integrity (PMI), DNA fragmentation index (DFI) and semen quality after storage at 5 °C for 24 hours. TSN and DFI were higher for protocol A as compared to protocol B. TSN in stallions submitted to protocol A (1st ejaculate) was influenced by season, whereas TSN remained unchanged in stallions submitted to protocol B. PM exceeded 70 % in every ejaculate and the protocol did not significantly affect motility. PMI was lower in ejaculates of 33.4 months old stallions on average as compared to older individuals. No other semen parameter was affected by the stallions' age. In conclusion, breeding soundness examination in 33.4 months old stallions is feasible and semen analysis after daily collection for 7 days provides less variable results throughout the year than semen analysis with protocol A. Therefore, semen analysis after daily collection for 7 days is recommended for young stallions. [ABSTRACT FROM AUTHOR]

Although female preferences are well studied in many mammals, the possible effects of the oestrous cycle are not yet sufficiently understood. Here we investigate female preferences for visual and non-visual male traits relative to the periodically cycling of sexual proceptivity (oestrus) and inactivity (dioestrus), respectively, in the polygynous horse (Equus caballus). We individually exposed mares to stallions in four experimental situations: (i) mares in oestrus and visual contact to stallions allowed, (ii) mares in oestrus, with blinds (wooden partitions preventing visual contact but allowing for acoustic and olfactory communication), (iii) mares in dioestrus, no blinds, and (iv) mares in dioestrus, with blinds. Contact times of the mares with each stallion, defined as the cumulative amount of time a mare was in the vicinity of an individual stallion and actively searching contact, were used to rank stallions according to each mare’s preferences. We found that preferences based on visual traits differed significantly from preferences based on non-visual traits in dioestrous mares. The mares then showed a preference for older and larger males, but only if visual cues were available. In contrast, oestrous mares showed consistent preferences with or without blinds, i.e. their preferences were mainly based on non-visual traits and could not be predicted by male age or size. Stallions who were generally preferred displayed a high libido that may have positively influenced female interest or may have been a consequence of it. We conclude that the oestrous cycle has a significant influence on female preferences for visual and non-visual male traits in the horse. [ABSTRACT FROM AUTHOR]

A high quality of stallion semen is of particular importance for maximum reproductive efficiency. In the present study, we estimated the relationships among estimated breeding values (EBVs) of semen traits and EBVs for the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) for 100 German Warmblood stallions using correlation and general linear model analyses. The most highly correlated sperm quality trait was total number of progressively motile sperm ( r = 0.36). EBV-PAT was considered in three classes with stallions 1 SD below (<80), around (80–120), and above (>120) the population mean of 100. The general linear model analysis showed significant effects for EBVs of all semen traits. EBVs of sperm quality traits greater than 100 to 110 were indicative for EBV-PAT greater than 120. Recommendations for breeding soundness examinations on the basis of the assessments of sperm quality traits and estimation of breeding values seem to be an option to support breeders to improve stallion fertility in the present and future stallion generation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Sperm from different stallions exhibit great differences in cryosurvival rates, which likely can be attributed to differences in cellular properties, such as membrane stability towards osmotic stress. The aim of this study was to obtain insights in factors determining membrane stability as well as cryosurvival. Pre-freeze osmotic tolerance was evaluated for individual stallions, and it was found that this cannot be used as a parameter to predict sperm cryosurvival rates. [ABSTRACT FROM AUTHOR]

Abstract: Stallion fertility is of high economic importance for the horse industry. The discovery of molecular mechanisms affecting fertility will be facilitated by the horse genome assembly, the development of genome-wide transcript and single-nucleotide polymorphism (SNP) arrays, and next-generation sequencing of whole mammalian genomes for analyzing complex fertility traits. Candidate genes can be identified via their involvement in key processes of horse reproduction or other species and their fertility-influencing mutations in genes described for species other than horses. We prioritized candidate genes that have been shown to play a major role in stallion reproduction and for which a relevant role in male reproduction in man or mice has been revealed, and located these genes on the horse genome assembly EquCab2.0. First, we sequenced candidate genes for stallion reproduction, including CRISP3, SPATA1, INHBA, ACE, SP17, FSHB, and PRLR, and screened these genes for polymorphisms associated with pregnancy rate per estrus in stallions. Genome-wide association studies using the equine Illumina 50K beadchip were performed to discover highly associated SNPs for stallion fertility. Among the candidate genes identified, we could find associations for candidate genes not yet analyzed. Molecular genetic studies of candidate genes from known pathways are encouraging, and with the novel tools available for genome-wide association studies and whole-genome sequencing, our knowledge on genetics of stallion fertility will be advanced. [Copyright &y& Elsevier]

Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures, from 30-10°C, whereas freezing-induced dehydration of the cells causes a more severe phase transition to a highly ordered gel phase. Sperm from individual stallions showed significant differences in post-thaw progressive motility, percentages of sperm with abnormal cell morphology, and chromatin stability. The biophysical membrane properties evaluated in this study, however, did not show clear differences amongst stallions with differences in sperm freezability. Cyclodextrin treatment to remove cholesterol from the cellular membranes increased the cooperativity of the suprazero phase transition, but had little effects on the subzero membrane phase behavior. In contrast, freezing of sperm in the presence of protective agents decreased the rate of membrane dehydration and increased the total extent of dehydration. Cryoprotective agents such as glycerol decrease the amount of energy needed to transport water across cellular membranes during freezing. [ABSTRACT FROM AUTHOR]

Stallion fertility is of high economic importance for the horse industry. The discovery of molecular mechanisms affecting fertility will be facilitated by the horse genome assembly and the development of novel tools for analysing complex genetic traits. Genetic markers in candidate genes, such as CRISP3, SPATA1 and INHBA, in breeding stallions have been associated with pregnancy rate per oestrus in mares. This paper reviews candidate autosomal, X and Y genes for stallion fertility, including genes encoding hormones and their receptors of the hypothalamic-pituitary axis, proteins of the seminal plasma, proteins involved in spermatozoa-ovum binding and genes influencing sexual development, as well as Y-specific genes. Their chromosomal location and gene structure are described, based on the horse genome assembly EquCab2.0 and a resource for markers located within or in close vicinity to the candidate genes (including pre-designed primer sequences). The application of genetic markers in improving stallion fertility for breeding and management is discussed. [ABSTRACT FROM AUTHOR]

Abstract: The reproductive efficiency of stabled domestic stallions is often lower than what could be expected from observations in feral herds. In the wild, stallions typically live with mares in harem bands, with other stallions in bachelor bands, or occasionally in mixed-sex transitional bands. We, therefore, argue that permanent contact with mares may increase reproductive efficiency of stallions suffering from low libido and/or fertility. We also provide a summary of our present knowledge of natural conditions, management, and husbandry of domestic stallions, and of intra- and intersexual behavioral interactions in horses. [Copyright &y& Elsevier]

Abstract: Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E Lp, Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH2 stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H2O-bending and -libration combination band, which yielded higher values for both E Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition. [ABSTRACT FROM AUTHOR]

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. • a dual-syringe pump system facilitates droplet formation for sperm vitrification. • a model is presented on droplet temperature profiles during solid surface vitrification. • Small droplet sizes and high contents of cryoprotective agents facilitate vitrification. • Sperm concentration negatively correlates with percentages of membrane intact sperm. • Sperm motility is lost after vitrification with high contents of cryoprotective agents. [ABSTRACT FROM AUTHOR]

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. • Shipment of frozen sperm on dry ice does not alter sperm viability and fertility. • PVP-40 and Ficoll-70 inhibit ice recrystallization during frozen storage. • Sperm samples need to be cooled below −40 °C before they can be plunged in LN2. • Addition of synthetic polymers increases stability under non-ideal conditions. [ABSTRACT FROM AUTHOR]

We performed a genome-wide association study for semen quality traits in 139 German Warmblood stallions. Stallions were genotyped using the Illumina equine SNP50 Beadchip. Traits analysed were de-regressed estimated breeding values (EBVs) for gel-free volume, sperm concentration, total number of sperm, progressive motility and the total number of progressively motile sperm. The GWAS revealed 29 SNPs on 12 different chromosomes as genome-wide significantly associated with semen quality traits. For ten genomic regions we could retrieve candidate genes influencing stallion fertility. Among the candidate genes, we could find the genes encoding cysteine-rich secretory proteins ( CRISP1 , CRISP2 and CRISP3 ). This was the first GWAS in horses performed for semen quality traits. [ABSTRACT FROM AUTHOR]

Abstract: Density gradient centrifugation can be used for selection of sperm of superior quality and removal of seminal plasma for use in artificial insemination. In this study, the use of two-layer iodixanol density gradient centrifugation was evaluated for processing of stallion semen. The protocol includes centrifugation through a 16% iodixanol top layer of 1.090gmL−1 and collection of motile and intact sperm on a 30% iodixanol bottom layer of 1.165gmL−1. Sperm recovery and effects on sperm quality were determined during cold storage as well as after cryopreservation and compared with ordinary dilution and centrifugation. Two-layer iodixanol density gradient centrifugation allows for selection of greater percentages of morphologically normal and progressively motile sperm compared to ordinary centrifugation. This likely results from collecting sperm on the bottom layer that functions as cushion fluid, which prevents mechanical forces as occur when sperm are packed in a pellet. In addition, percentages of membrane and chromatin integrity are increased when cells are selected based on their density via centrifugation through the top and bottom layers. Removal of seminal plasma and increased initial percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater percentages of progressively motile and membrane intact sperm during cold storage as well as after freezing and thawing. In conclusion, the two-layer iodixanol density gradient centrifugation protocol described in this manuscript allows for selection of stallion sperm with greater survival rates for cold storage and cryopreservation. [Copyright &y& Elsevier]

Abstract: The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg−1 already results in an increase in plasma membrane damaged cells, increasing up to 50% at an osmolality of 136 mOsm kg−1. Plasma membrane damaged stallion sperm do not show an osmotic response after 10 min incubation in hypotonic conditions, and their volume is smaller as compared to viable cells. It is shown that inclusion or exclusion of different subpopulations greatly affects Boyle van ''t Hoff behavior and therewith determination of the osmotic inactive volume. Osmotic inactive volumes were determined to be 76% and 46% of the isotonic volume for the whole sperm population and the plasma membrane intact viable cells, respectively. In addition, viable subpopulations with different average cell volumes also show different osmotic behavior. The main subpopulation of viable cells increased up to 1.6 times its isotonic volume upon exposure to 150 mOsm kg−1, and exhibited an osmotic inactive volume of 79%. [Copyright &y& Elsevier]

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. • Stallion sperm viability is preserved at 4 °C in barium-alginate core-shell structures. • Viability of encapsulated sperm is lower as compared to diluted sperm. • Calcium-alginate encapsulation negatively affects stallion sperm motility. • Encapsulation in solid alginate matrix negatively impacts stallion sperm viability. [ABSTRACT FROM AUTHOR]

Effects of a plasmolysed yeast product enriched with herbs, malt, honey and orange syrup on semen characteristics and oxidative status in stallions were evaluated. Twenty stallions (mean age ± standard deviation = 9.5 ± 4.5 years) were randomly divided into a treatment group (n = 10) receiving 0.06 mL/kg bodyweight of plasmolysed herbal yeast, and a control group (n = 10) receiving the same amount of placebo daily in the feed for 10 weeks. Ejaculates were collected weekly from all stallions starting at Week 0. Volume, sperm concentration, motility, and velocity were evaluated immediately, 24 and 48 h after cooled storage at 5 °C. At the two storage time points, membrane lipid peroxidation was determined using the BODIPY-C 11. Additionally, blood samples were collected at Weeks 0, 1, 5 and 9, and analysed for antioxidant status, consisting of superoxide dismutase, cholesterol, thiobarbituric acid reactive substances, and non-esterified fatty acids. Due to the nature of the data, the Mann-Whitney U test was applied as preliminary analysis. The BODIPY-C 11 in the semen was less at 24 h and greater at 48 h after collections in Week 1 to 3 (P < 0.01) and Week 1 to 10 (P < 0.05) compared with Week 0 in the treatment compared to control group. There were no significant differences between groups for all values for other seminal and blood variables evaluated. In conclusion, feed supplementation with plasmolysed herbal yeast temporarily improved the antioxidant status of stallion semen, which might be of benefit for preservation of cooled semen. [ABSTRACT FROM AUTHOR]

Success rates for cryopreservation of equine sperm show a large variation among individual stallions. Factors that have been implicated with this inter-male variation in cryosurvival are the ability of sperm to tolerate osmotic stress, and temperature-induced changes in membrane phase state and organization. Cells are exposed to osmotic stress both upon the addition of cryoprotective agents as well as during freezing and thawing. Moreover membranes undergo phase changes during cooling and warming causing transport of molecules that normally are membrane-impermeable. The aim of this study was to evaluate intra-individual variability in osmotic properties of stallion sperm and its correlation with cryosurvival. Furthermore, the temperature dependency of hypo-osmotic tolerance was studied and correlated with membrane phase behavior. Pre- and post-freeze sperm motility and plasma membrane integrity were assessed using computer assisted sperm analysis and flow cytometry. The critical osmolality, at which half of the sperm population survived exposure to hypotonic saline solution, was taken as a measure for osmotic tolerance. Temperature-dependent changes in membrane conformational disorder were evaluated using Fourier transform infrared spectroscopy. Sperm membranes display a main phase transition in the 10–30°C temperature range, with two distinct melting events. The melting event around 30°C becomes more pronounced with removal of cholesterol. 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. The decreased tolerance towards exposure to hypotonic stress at 4°C could be caused by the membrane phase transition which the membranes undergo during cooling. The decreased hypo-osmotic tolerance at 37°C might be due to the fluidity of stallion sperm membranes at this temperature. The critical osmolality at 22°C, was found to vary between 55 and 170mOsmkg−1 among sperm from different stallions. Clear correlations were found for pre- versus post-freeze sperm motility as well as membrane integrity, assessed in either freezing extender or after dilution in isotonic medium. Pre-freeze percentages of membrane intact sperm after exposure to hypotonic stress showed a correlation with sperm motility after cryopreservation. This correlation, however, disappeared when data were corrected for initial numbers of membrane intact sperm in the sample. It is concluded that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions. Furthermore, membrane phase behavior and the overall membrane fluidity at a given temperature are involved in osmotic stability. Source of funding: This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflict of interest: None declared. harriette.oldenhof@tiho-hannover.de [ABSTRACT FROM AUTHOR]