Enrichment of thawed boar spermatozoa with an intact membrane using Magnetic Activated Cell Sorting.

Not all boar sperm samples survive cryopreservation well. A method of eliminating damaged sperm might enable more cryopreserved boar semen to be used for pig breeding. In this study we investigated the use of Magnetic Activated Cell sorting (MACS) to eliminate damaged sperm from thawed boar semen samples. The thawed samples were mixed with Dead cell removal particles and were applied to the column in a SuperMACS II. Different fractions were collected: Original sample (O), Flow-through (FT), and Eluate (E). Sperm membrane integrity, mitochondrial membrane potential and reactive oxygen species were evaluated by flow cytometry after staining with SYBR 14 and propidium iodide, or 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide, or hydroethidine and dichlorodihydrofluorescein diacetate, respectively. The FT samples had increased membrane integrity, a greater proportion of sperm with high mitochondrial membrane potential and a greater proportion of sperm negative for hydrogen peroxide than O samples (P<0.0001), which in turn had increased membrane integrity than E samples (P <0.0001). However, differences were seen between boars. The FT samples had increased values of live, superoxide positive sperm than O samples (P <0.0001) and O samples had greater values than E samples (P <0.0001), while there was no effect of boar. Sperm quality was best in the FT fraction, comprising approximately 32% of the sperm sample. In conclusion, although there were differences between boars, MACS separation can improve sperm quality in thawed semen samples. It would be interesting to see if this improvement is reflected in fertility outcomes.

A combination of biomarkers for predicting stallion sperm fertility.

Equine breeding would benefit greatly from reliable biomarkers of stallion or ejaculate fertility. The aim of the study was to investigate how several in vitro sperm characteristics correlate with fertility after artificial insemination, to explore the potential to build a fertility prediction model for stallions. Cooled insemination doses (3-5 per stallion) were obtained from various studs. Sperm membrane integrity, acrosome integrity, chromatin integrity, mitochondrial membrane potential, and reactive oxygen species production were evaluated by flow cytometry 24-30 h after semen collection, and sperm motility was assessed by computer aided sperm analysis. Calcein violet was used to differentiate viable spermatozoa. Per season pregnancy rates for these stallions were available the following year. Positive correlations were found between pregnancy rate and straightness (r = 0.43, p ≤ 0.001), as well as pregnancy rate and the proportion of living hydrogen peroxide positive spermatozoa (r = 0.32, p ≤ 0.05). There were negative correlations between pregnancy rate and amplitude of lateral head displacement (r = -0.26, p ≤ 0.05), and between pregnancy rate and the mean fluorescence of dead superoxide positive spermatozoa (r = -0.46, p < 0.001). Principal component analysis indicated that motility, membrane integrity, DNA fragmentation, and reactive oxygen species production were associated with pregnancy rate. Therefore, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates. However, data from more individuals would be required to construct a model for fertility prediction.

Single layer centrifugation as a method for bacterial reduction in bull semen for assisted reproduction.

Semen samples contain bacteria originating from the animal urogenital tract, environment, and/or contamination during semen processing, negatively affecting sperm quality by producing toxins and/or competing for nutrients in extenders. The aims of this study were to evaluate two methods of Single-layer centrifuges (SLC), high and low density colloid, as a method for bacterial removal from bull semen, and to evaluate sperm quality after treatment. In total, semen samples from 20 bulls (3 ejaculates per bull) were used in this study. Bacterial reduction was evaluated by bacterial quantification (colony forming unit - CFU/mL) while bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after culturing bacteria on blood agar. Sperm motility parameters were evaluated by Computer Assisted Sperm Analyses (CASA), and sperm chromatin structure assay (SCSA) by Flow cytometry. Both, High and Low density SLC reduced number of bacteria significantly (p < 0.001) compared with control. The difference in bacterial count between High and Low SLC was also significant (p < 0.001). Furthermore, High density SLC was successful in removing almost all Bacillus and Proteus spp. Most CASA parameters were significantly improved after both treatments (p < 0.001, p < 0.01, p < 0.05). The Deoxyribonucleic acid (DNA) fragmentation index evaluated by SCSA in High (p < 0.01) and Low (p < 0.05) SLC group differed significantly compared with control. Single-layer centrifugation (SLC) with either a high or a low density colloid is a suitable method for bacterial removal in bull semen.

Evaluation of domestic animal sperm head morphology via flow cytometric DNA labelling and pulse shape analysis using bull and stallion spermatozoa as model species.

The aim of the present study was to test a rapid, robust flow cytometric technique for the detection of sperm head abnormalities of domestic bulls and stallions. The so-called PulSA approach detects the pulse profiles of propidium-iodide labelled spermatozoa. In the first experiment, species-specific threshold values were established on sperm samples that were tested for sperm head abnormalities with a classic visual morphology analysis. In the second experiment, serial mixtures of bull and stallion spermatozoa mimicking different percentages of sperm head abnormalities were analysed. Non-metric multidimensional scaling showed a clear separation between the normal and mixed samples. The PulSA approach may be a useful tool in identifying sub- or infertile breeding males as well as in studying the evolutionary aspects of sperm morphology and morphometry.

Effect of Some Plant-Based Substances on Microbial Content and Sperm Quality Parameters of Bull Semen.

The rapid emergence of antibacterial resistance requires alternatives to antibiotics to be found, including for semen preservation. One of the possible alternatives would be to use plant-based substances with known antimicrobial effects. The objective of this study was to test the antimicrobial effect of pomegranate powder, ginger, and curcumin extract in two concentrations on bull semen microbiota after exposure for <2 h and 24 h. An additional aim was to evaluate the effect of these substances on sperm quality parameters. The bacterial count in semen was low from the beginning; however, a reduction was present for all tested substances compared with control. A reduction in bacterial count in control samples was also observed with time. Curcumin at a concentration of 5%, reduced bacterial count by 32% and was the only substance that had a slight positive effect on sperm kinematics. The other substances were associated with a decline in sperm kinematics and viability. Neither concentration of curcumin had a deleterious effect on sperm viability parameters measured by flow cytometry. The results of this study indicate that curcumin extract at a concentration of 5% can reduce the bacterial count and does not have a negative influence on bull sperm quality.

Deciphering sperm chromatin properties to predict stallion sperm fertility.

Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sciences. Aliquots of semen were stained for flow cytometry with acridine orange for the Sperm Chromatin Structure Assay (DNA fragmentation Index, %DFI), with chromomycin A3 (CMA) for protamine deficiency, and with monobromobimane (mBBr) for detection of total and free thiols and disulfide bonds. Per season pregnancy rates after insemination were obtained. Mixed linear models were used to analyze data. Negative correlations were found between pregnancy rate and %DFI (r = -0.35, P < 0.03) and pregnancy rate and free thiols (r = -0.60, P < 0.0001). Furthermore, there were positive correlations between total thiols and disulfide bonds (r = 0.95, P < 0.0001), and protamine and disulfide bonds (r = 0.4100, P < 0.01986). Since chromatin integrity, protamine deficiency and packaging were all associated with fertility, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates.

Sperm Quality in Young Bull Semen Can Be Improved by Single Layer Centrifugation.

Interest in using semen from young bulls is increasing due to identifying promising animals by genomic selection. However, sperm quality in these ejaculates may not reach currently accepted standards for the cattle breeding industry. The purpose of this study was to determine if centrifugation of semen from young bulls through the Bovicoll colloid could improve sperm quality sufficiently for the frozen semen to be acceptable for artificial insemination. Ejaculates from 19 young bulls were split and either processed by Single-Layer Centrifugation (SLC) or not (CON) before freezing. After thawing, sperm quality was evaluated by determination of membrane integrity, mitochondrial membrane potential, DNA integrity, production of reactive oxygen species, sperm morphology and motility. Approximately half of the CON samples reached acceptable post-thaw quality (membrane integrity ≥ 40%) despite being below the breeding company´s desired sperm concentration threshold pre-freezing. In the remaining samples, sperm quality was improved by SLC such that 45% of them reached acceptable quality post-thaw. Almost 75% of the young bull sperm samples could have produced usable frozen semen doses by adjusting the breeding company´s current processing protocols. Since lowering the generation interval has a direct effect on the genetic gain per year, SLC could aid genetic progress in cattle breeding.

Influence of Single Layer Centrifugation with Canicoll on Semen Freezability in Dogs.

This study evaluated how semen selection by single layer centrifugation (SLC) with Canicoll affects semen freezability in dogs. A total of eighteen ejaculates, collected from dogs with optimal and suboptimal semen quality (optimal: normal morphology (NM) ≥ 80%, n = 9; suboptimal: NM between 60 and 79%, n = 9), were divided into two aliquots and subjected to standard centrifugation or SLC before cryopreservation. Motility, NM, membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were improved in fresh samples after SLC, regardless of semen quality, but at the expense of some good quality spermatozoa. After thawing, NM and membrane integrity were improved in SLC-selected semen in both semen qualities. Interestingly, MMP was also higher but only in optimal quality semen. Still, spermatozoa from suboptimal quality semen did not survive freezing to the same extent as spermatozoa from optimal quality semen, even after selecting superior spermatozoa. Semen selection with Canicoll is, therefore, an effective technique to isolate a subpopulation of high-quality spermatozoa and obtain sperm samples of better quality after thawing, but is not sufficient to improve the intrinsic inferior freezability of suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile.

Post-thaw semen quality in young bull ejaculates before being accepted for commercial semen doses.

BACKGROUND: Genomic selection enables bulls with desirable characteristics to be identified at a young age, but sperm quality can be poor in the ejaculates of young bulls. Few studies have been done on post-thaw sperm quality in bulls less than 10 months old. The objective of this study was to determine the age at which post-thaw sperm quality was acceptable for artificial insemination. METHODS: Semen was collected by artificial vagina; samples containing 100-500 million spermatozoa/ml were frozen for this study. Post-thaw analyses of membrane integrity (MI), mitochondrial membrane potential (MMP), chromatin integrity, morphology, production of reactive oxygen species and sperm kinematics were made. RESULTS: The age at which ejaculates exceeded the breeding company's thresholds of acceptance varied considerably among individuals, with 285 days being the earliest. Morphology (p < 0.003), MI (p = 0.0096), high MMP (p = 0.043) and superoxide production (p = 0.0084) increased between the first and last ejaculates but reached acceptable levels at different ages for individual bulls. CONCLUSIONS: It was possible to obtain acceptable post-thaw sperm quality from samples even though sperm concentration was lower than the breeding company's threshold. Therefore, it might be feasible to use ejaculates earlier than is currently considered possible, by modifying semen handling protocols.

Practical Method for Freezing Buck Semen.

Although several protocols for cryopreserving buck semen are described in the literature, they differ widely in factors such as season and method of semen collection, extender and sperm concentration. Therefore, choosing a protocol that is suitable for a particular on-farm situation can be problematic. In the present study, semen was collected by artificial vagina from seven bucks on a farm located approximately 90 minutes' drive away from the laboratory, about 6 weeks before the start of the goat breeding season. The semen was immediately extended in warm semen extender containing soy lecithin and was placed in an insulated box with a cold pack for up to 4 h, during semen collection from the remaining bucks and subsequent transport to the laboratory. Following centrifugation at 4 °C and resuspension in the soy lecithin extender to a sperm concentration of 800 × 106 spermatozoa/mL, 0.25 mL plastic straws were filled and frozen in racks 4 cm above the surface of liquid nitrogen. This simple protocol resulted in an acceptable post-thaw quality for all seven bucks, with a mean post-thaw motility of 55 ± 21% and mean fragmented chromatin of 3.27 ± 1.39%. Normal sperm morphology was >90% in all ejaculates. The semen was sent to a gamete bank for long-term storage.

Variation among stallions in sperm quality after single layer centrifugation.

Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48 hr using computer-assisted sperm analysis and flow cytometry. Sperm quality was better in SLC than in CON at all timepoints, especially chromatin integrity and MI (p < .0001 for both), and some categories of ROS production (e.g. proportion of live hydrogen peroxide negative spermatozoa, p < .0001), but the degree of improvement varied among stallions and type of ROS (p < .05-p < .0001). Total and progressive motility were also better in SLC samples than in CON at 24 and 48 hr (p < .0001), although the effect on sperm kinematics varied. The interaction of treatment, time and stallion was not significant. In conclusion, sperm quality was better in SLC samples than in CON, although there was considerable individual variation among stallions. The improvement in sperm quality, particularly in chromatin integrity, was clearly beneficial, and therefore the use of this technique would be warranted for all stallion semen samples.

Cryopreservation of dog semen in a Tris extender with two different 1% soybean preparations compared with a Tris egg yolk extender.

Egg yolk is widely used as a cryoprotectant in dog semen extenders, but there is a risk of contamination with animal pathogens. In addition, egg yolk may vary in composition, making it difficult to standardize the extender. Lecithin is an animal protein-free alternative to egg yolk for semen cryopreservation. Recently, it was shown that 1% of soybean lecithin type II-S was better than 2% for freezing canine semen. The aim of the study was to compare two different types of soybean lecithin, with egg yolk as a control. Ejaculates from eight dogs were divided into three equal parts and diluted with a Tris-based extender, containing either 20% egg yolk, 1% soybean lecithin Type II-S or 1% soybean lecithin Type IV-S. The samples were then frozen. Sperm motility was evaluated by computer-assisted sperm analysis (CASA), acrosome integrity (FITC-PNA/PI) and sperm membrane integrity (SYBR-14/PI) post-thaw, as well as after 2 and 4 hr incubation at 37°C. Post-thaw sperm chromatin structure assay and plasma membrane integrity were evaluated by flow cytometry. Total motility, sperm plasma membrane integrity and acrosome integrity were significantly better in the egg yolk extender than in the two soybean lecithin-based extenders. Individual motility post-thaw differed more than in the fresh samples, illustrating individual differences in tolerance to the cryostress. The DNA Fragmentation Index (% DFI) was significantly lower in the Tris egg yolk (TEY) extender compared to any of the soybean-based extenders. The number of high green stained spermatozoa were significantly higher in Type IV-S compared to the control TEY extender. In conclusion, egg yolk was superior to the two lecithin-based extenders to cryopreserve canine semen.

Matrix metalloproteinase (MMP)-2, MMP-9, semen quality and sperm longevity in fractionated stallion semen.

Matrix metalloproteinase (MMP)-2 and MMP-9 are gelatinases that take part in several reproductive processes. The aim of this study was to measure levels of MMP-2 and MMP-9 in fractionated stallion ejaculates, and to evaluate the association between these components and semen quality, and sperm longevity during cooled storage. Semen quality were assessed separately for sperm-rich fractions (HIGH), sperm-poor fractions (LOW), and whole ejaculate samples (WE) from 33 stallions. After cooled storage with SP either present or removed, sperm motility and DFI were determined. The relative activity of the pro-form of MMP-2, active MMP-2 and total MMP-9 were evaluated using gelatin zymography, and all were present in all fractions of the stallion's ejaculate, with higher relative activity of the latent than active forms and the highest relative activity in the HIGH fraction. The relative activities of MMP-2 and MMP-9 were positively correlated to sperm concentration and total sperm count, but only in the HIGH fraction and not in LOW or WE. The relative activities of MMPs were not related to differences in sperm longevity during cooled storage, measured as sperm motility and DFI. There was a harmful effect of SP on DFI during storage, but this effect was not associated with differences in the relative activities of MMPs. In conclusion, the relative activities of MMPs are not useful as markers for semen quality (other than sperm concentration), or sperm survival during storage in horses.

Sperm chromatin integrity and DNA methylation in Norwegian Red bulls of contrasting fertility.

In this study, the complexity of chromatin integrity was investigated in frozen-thawed semen samples from 37 sires with contrasting fertility, expressed as 56-day non-return rates (NR56). Protamine deficiency, thiols, and disulfide bonds were assessed and compared with previously published data for DNA fragmentation index (DFI) and high DNA stainability (HDS). In addition, in vitro embryo development and sperm DNA methylation were assessed using semen samples from 16 of these bulls. The percentages of DFI and HDS were negatively associated with NR56 and cleavage rate and positively associated with sperm protamine deficiency (p < 0.05). Significant differences in cleavage and blastocyst rates were observed between bulls of high and low NR56. However, once fertilization occurred, further development into blastocysts was not associated with NR56. The differential methylation analysis showed that spermatozoa from bulls of low NR56 were hypermethylated compared to bulls of high NR56. Pathway analysis showed that genes annotated to differentially methylated cytosines could participate in different biological pathways and have important biological roles related to bull fertility. In conclusion, sperm cells from Norwegian Red bulls of inferior fertility have less compact chromatin structure, higher levels of DNA damage, and are hypermethylated compared with bulls of superior fertility.

Assessment of source and treated water quality in seven drinking water treatment plants by in vitro bioassays - Oxidative stress and antiandrogenic effects after artificial infiltration.

Drinking water quality and treatment efficacy was investigated in seven drinking water treatment plants (DWTPs), using water from the river Göta Älv, which also is a recipient of treated sewage water. A panel of cell-based bioassays was used, including measurements of receptor activity of aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), peroxisome proliferator-activated receptor alpha (PPARα) as well as induction of oxidative stress (Nrf2) and micronuclei formation. Grab water samples were concentrated by solid phase extraction (SPE) and water samples were analyzed at a relative enrichment factor of 50. High activities of AhR, ER and AR antagonism were present in WWTP outlets along the river. Inlet water from the river exhibited AhR and AR antagonistic activities. AhR activity was removed by DWTPs using granulated activated carbon (GAC) and artificial infiltration. AR antagonistic activity was removed by the treatment plants, except the artificial infiltration plant, which actually increased the activity. Furthermore, treated drinking water from the DWTP using artificial infiltration exhibited high Nrf2 activity, which was not found in any of the other water samples. Nrf2 activity was found in water from eight of the 13 abstraction wells, collecting water from the artificial infiltration. No genotoxic activity was detected at non-cytotoxic concentrations. No Nrf2 or AR antagonistic activities were detected in the inlet or outlet water after the DWTP had been replaced by a new plant, using membrane ultrafiltration and GAC. Neither target chemical analysis, nor chemical analysis according to the drinking water regulation, detected any presence of chemicals, which could be responsible of the prominent effects on oxidative stress and AR antagonistic activity in the drinking water samples. Thus, bioanalysis is a useful tool for detection of unknown hazards in drinking water and for assessment of drinking water treatments.

Single Layer Centrifugation with 20% or 30% Porcicoll separates the majority of spermatozoa from a sample without adversely affecting sperm quality.

Centrifugation of boar semen through one layer of 40% colloid (Porcicoll) was previously shown to separate spermatozoa from bacteria without having a detrimental effect on sperm quality. However, some spermatozoa were lost. The purpose of the present study was to determine whether 20% or 30% Porcicoll could be used to recover most of the spermatozoa without impacting on sperm quality. Insemination doses (n = 10) from a commercial boar station were sent to the laboratory at the Swedish University of Agricultural Sciences and processed by Single Layer Centrifugation with 20% and 30% Porcicoll approximately 7 hr after semen collection. The resulting sperm samples and controls were evaluated for sperm quality immediately and again after storage at 16-18°C for 4 and 7 days. Sperm recovery was 94 ± 18% and 87 ± 15% for 20% and 30% Porcicoll, respectively (p > .05). Sperm mitochondrial membrane potential and chromatin integrity were unaffected (p > .05). The proportion of live spermatozoa producing superoxide (9 ± 8%, 7 ± 6% and 3 ± 1%; p < .05), and the proportion of spermatozoa with high stainability DNA (0.68 ± 19%, 0.61 ± 0.22% and 0.96 ± 0.23%; p < .05- <0.01), were marginally increased whereas membrane integrity, although high, was lower in the centrifuged samples than in the controls (82 ± 8%, 83 ± 5% versus 92 ± 4%; p < .05). In conclusion, centrifugation through 20% or 30% Porcicoll enables most spermatozoa to be recovered, without having a major effect on sperm quality. These results are encouraging for further studies involving microbiological investigation of the processed samples, and scaling-up to process larger volumes of boar ejaculates.

Effects of cashew gum and nanoparticles on cooled stallion semen.

BACKGROUND: Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes at 4 °C. RESULTS: Aliquots were analysed by computer-assisted sperm motility analysis on the day of collection, and after 24 h and 48 h of cold storage. After 48 h, the total motility with NP1 (78.53 + 6.31%) was similar to control 85.79 + 6.31% at 0 h. The same pattern was observed for progressive motility. Membrane integrity assessed by flow cytometer was similar between control, TOC and G1 at all storage times. The DNA fragmentation in the control group increased at all time points, whereas chromatin integrity was maintained after 24 h in TOC and NP0.5 compared to 0 h. There was no increase in the proportion of live spermatozoa producing hydrogen peroxide, but there was a tendency for an increased proportion of spermatozoa in the live superoxide category in CG1 after 24 h cooled storage. CONCLUSIONS: The addition of CG or CG-derived NP to extender for stallion semen was not harmful to the sperm cells.

Effects of season and single layer centrifugation on bull sperm quality in Thailand.

OBJECTIVE: The aim of study was to investigate the effects of season and single layer centrifugation (SLC) before cryopreservation on post-thaw bull sperm quality in Thailand. METHODS: Semen was collected from 6 bulls (Bos indicus) in summer, rainy season and winter 2014 through 2016. Semen characteristics, sperm morphology, sperm kinematics, viability, chromatin structure and mitochondrial membrane were evaluated. Meteorological data were available from the local meteorological station. RESULTS: Season had an effect on semen characteristics in the raw ejaculate, with higher proportions of normal spermatozoa and lower abnormalities in winter than in the other two seasons. Sperm kinematics, viability, DNA fragmentation index, and mitochondrial membrane potential were not different between seasons. Sperm samples selected by SLC had greater normal morphology and a lower proportion with bent tails than controls and higher values of progressive motility (PRO), beat cross frequency, linearity, straightness, wobble (WOB), and lower values of slow motility, velocity average path (VAP), velocity curved line, and amplitude of lateral head displacement than controls. In addition, SLCselection had a favorable effect on PRO, VAP, and WOB that differed among seasons. CONCLUSION: Our results suggested that these bulls were well adapted to their location, with season having an effect on sperm morphology. Moreover, SLC could be used prior to cryopreservation, regardless of season, to enhance normal morphology and kinematics of bull sperm samples without adversely affecting other parameters of sperm quality. However, there was considerable variation among bulls in DNA fragmentation index, mitochondrial membrane potential and sperm viability. In addition, SLC had a positive effect on sperm morphology and sperm kinematics, which could be expected to influence fertility.

Season does not have a deleterious effect on proportions of stallion seminal plasma proteins.

The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of non-heparin binding proteins as well as of heparin-binding proteins was found. Analysis of semen characteristics revealed a somewhat contrasting picture. While only minor variations in sperm kinematics and sperm morphology were found between seasons, the flow-cytometric measurements of mitochondrial membrane potential and also, to some extent, reactive oxygen species production indicated lower sperm quality in the breeding season. Chromatin integrity and testosterone levels were unchanged between seasons. The results suggest that stallion ejaculates could be used year-round for freezing, since only minor differences in protein composition exist between the breeding and non-breeding seasons, as well as between good and bad freezers. In addition, sperm quality is not impaired during the non-breeding season.

Upregulation of CRISP-3 and kallikrein in stallion seminal plasma is associated with poor tolerance of cooled storage.

For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expression in SP with either positive or negative effects on sperm survival during storage was studied using two-dimensional differential gel electrophoresis and liquid chromatography-mass spectrometry. Lower sperm chromatin integrity was associated with upregulation of the proteins kallikrein, CRISP-3 and HSP-1, while higher chromatin integrity was associated with upregulation of TIMP-2. In the sperm-rich fractions, kallikrein and CRISP-3 differed significantly between SP samples with differing effects on sperm chromatin integrity. In the sperm-poor fractions, TIMP-2 and HSP-1 differed significantly between the two SP groups. Differences in the seminal plasma proteome are associated with sperm longevity during cooled storage.