Freezability of stallion spermatozoa varies considerably between individuals. The aim was to investigate whether adding seminal plasma (SP) from good freezer or bad freezer stallions improved post-thaw sperm acrosomal integrity. Semen samples were available where SP-free spermatozoa from good freezer and bad freezer stallions prepared by SingleLayer Centrifugation (S) were treated with pooled SP from the same stallions prior to freezing. Control samples were frozen conventionally (C). Post-thaw acrosomal integrity was assessed by flow cytometry with/without stimulation by calcium ionophore. Higher proportions of live acrosome-intact spermatozoa (p = 0.04) and lower proportions of dead acrosome-intact spermatozoa (p = 0.48) occurred in non-stimulated S samples than in C. Acrosome integrity was not affected by either type of SP. However, in ionophore-stimulated samples, SP from good freezer stallions produced higher proportions of live acrosome-intact spermatozoa than C (p = 0.047), whereas SP from bad freezer stallions resulted in higher proportions of live acrosome-reacted spermatozoa than C (p = 0.04); the proportion of dead acrosome-intact spermatozoa was higher in C than in S (p = 0.05). Thus, S had a lower propensity to acrosome-react under stimulating conditions than C; the effect of adding SP depends on its origin. Further research is required to optimise cryopreservation protocols for stallion semen.

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.

IntroductionSince boar spermatozoa show a marked deterioration in sperm quality when cooled, insemination doses are usually stored at 16–18 °C. However, maintaining this temperature during transport of semen doses is challenging, particularly during the summer months. An alternative could be to store the doses at 4 °C if cold-shock to the sperm could be prevented. The objective of this study was to evaluate boar sperm quality and fertility in in vitro fertilization after storage in AndroStar Premium at 4 °C for 1 week.MethodsInsemination doses (n = 9) in AndroStar Premium from a commercial boar semen collection station were transported to the laboratory at approximately 20 °C. At the laboratory, sperm quality evaluation and was preformed and each dose was split; half of each ejaculate was stored in a climate-controlled box at 16–18 °C, the other was slowly cooled to 4 °C. Both samples were stored for 1 week before further sperm quality evaluation and in vitro fertilization (IVF) were performed. Mean values were tested using generalized linear regression, with treatment and boar as fixed factors; p ≤ 0.05 was considered significant.ResultsSperm membrane integrity (mean ± sem: 91 ± 0.05 and 83 ± 0.09% for 16 and 4 °C, respectively) and superoxide production (6.79 ± 2.37 and 13.54 ± 6.23% for 16 and 4 °C, respectively), were different between treatments. The DNA fragmentation index was lower in cold-stored samples than in conventionally stored samples (3.74 ± 2.25 and 7.40 ± 3.36% for 4 and 16 °C, respectively). The numbers of oocytes developing to blastocyst on Day 6 (mean ± sd: 9.0 ± 8.0 and 6.0 ± 5.0%, for storage at 16 and 4 °C, respectively) were not different between treatments.DiscussionTherefore, storage of boar semen doses in AndroStar Premium at 4 °C for up to 7 days would be a viable alternative to current praxis.

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.

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.

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.

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.

There are stallions with apparently good sperm quality that achieve low pregnancy rates when their semen is used for artificial insemination (AI). Thus, additional methods of evaluating sperm quality that result in better methods of predicting stallion fertility, are essential. Semen samples were obtained in April-June from 8 stallions, 4 trotters and 4 warmblood sport horses, which were sampled Monday, Wednesday and Friday each week. From each stallion, 1-4 samples were obtained, and evaluated after cold storage for 24 hours. The samples were evaluated for kinematic variables using CASA, and for membrane integrity, mitochondrial membrane potential (MMP), production of reactive oxygen species, acrosome integrity and chromatin integrity using flow cytometry. Relationships with fertility, calculated as number of pregnant mares/number of inseminated mares for samples used for artificial insemination after transport in cold storage, were evaluated by linear regression, following log-transformation of variables found not to be normally distributed. The following sperm variables were found to have a positive relationship with fertility: Straightness (r2=0.86, p= 0.0009), Viable with High mitosoxred/Low MMP (r2=0.70, p= 0.01), Viable non acrosome reacted (r2=0.68, p= 0.01), Viable by calceinviolet (r2=0.67, p= 0.01), Viable non H2O2-producing (r2=0.66, p= 0.01), Viable with High mitosoxred/High MMP (r2=0.65, p= 0.02), Viable non superoxide-producing (r2=0.60, p= 0.02), Linearity (r2=0.56, p= 0.03). The only variable found to have a negative relationship with fertility was Amplitude of lateral head displacement (r2=0.63, p= 0.02). The other 26 measured variables did not show a significant relationship with fertility. The results of the study shows that both kinematic variables as well as flow cytometric analysis can be valuable for relating fertility with sperm quality in vitro. The flow cytometry results indicate that analysis of the production of different reactive oxygen species can be valuable when predicting stallion fertility. Acknowledgments: Special thanks to Menhammar, Västerbo, Markebäck and Stockholm Seminstation for providing stallion semen. This work was supported by funding from FORMAS (2018-01083; awarded to AJ). The work was also supported by the Swedish University of Agricultural Sciences via the Cells for Life Platform.

In this study, the relationships between post-thaw bull sperm characteristics and hyperketonemic conditions after coincubation with cow plasma or media were determined to investigate if such a condition could affect bull sperm characteristics. Two experiments were conducted. In experiment 1, blood samples were collected from 31 cows to prepare plasma. Cows were independently categorized into two groups according to plasma β-hydroxybutyrate (BHB) concentrations (above or below 1.2 mM). Thawed bull semen was diluted and incubated with diluted plasma; motility parameters were evaluated using Computer Assisted Semen Analysis (CASA). In experiment 2, a pooled sample of thawed semen was diluted and divided into three aliquots: without BHB (control) and treated with either 1.2 mM (1.2) or 3 mM (3) BHB. In addition to motility, flow cytometric analyses were carried out. In experiment 1, the overall motility decreased significantly in plasma containing high (≥1.2 mM) BHB compared to plasma containing low (<1.2 mM) BHB. In experiment 2, the overall motility tended to be lower in BHB (3 mM)-supplemented samples. The supplementation of 3 mM BHB increased the proportion of live superoxide-positive sperm and sperm with high mitochondrial potential, while the DNA fragmentation index decreased.

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.

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.

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.

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.

Sperm selection techniques, such as magnetic activated cell sorting (MACS) and colloid centrifugation, are reported to select good quality spermatozoa from semen samples of various species. Although the sperm quality of fresh boar semen is usually good, cryopreservation has a negative effect on parameters such as plasma membrane integrity and mitochondrial activity. Therefore, the objective of the present study was to determine whether MACS or centrifugation through a single layer of colloid (Single Layer Centrifugation, SLC) would be beneficial in enriching thawed boar sperm samples for viable spermatozoa with active mitochondria and good chromatin integrity. Frozen samples from three boars, three ejaculates per boar, were thawed and split. One part was selected by MACS, one was prepared by SLC, and the remainder served as the control. Controls and the selected sperm samples were evaluated for sperm quality (plasma membrane integrity, chromatin integrity, mitochondrial membrane potential and production of reactive oxygen species). Although several aspects of sperm quality were improved in the SLC-selected sperm samples compared to control, the flow-through MACS samples were only improved in having a lower proportion of spermatozoa with immature chromatin (Hi green fluorescence) compared to the labeled control. Sperm quality in the SLC samples was better than in the flow-through samples from MACS. Therefore, despite promising reports of the use of MACS for selecting good quality spermatozoa from semen in other species, the method was not useful for improving sperm quality in the thawed boar sperm samples in this experiment.

Despite the relatively long history of captive breeding, the Arctic charr still exhibits a generally low, but highly variable reproductive performance in aquaculture. A recent publication exposed potential paternal factors influencing the reproductive outcome of Arctic charr broodstock from the Swedish breeding program. Interestingly, the paternal factor appeared to be more closely connected to embryo survival than to fertilisation rates. This lead to speculations on whether e.g. chromatin related issues, potentially related to oxidative stress could be involved. In order to investigate this hypothesis the present study assessed the levels of DNA fragmentation, using the SCD-method, and membrane integrity, using flow cytometry, in sperm of farmed Arctic charr. Moreover, the existence of associations was tested between DNA fragmentation and membrane integrity in individual semen samples and viability of their resulting progeny. We found high levels of DNA fragmentation in sperm from the Arctic charr sires, ranging from 24% to 86% with a median of 67%. Membrane integrity values were high, with individual levels of 93.1% to 99.6% viable sperm cells, median 98.8%. DNA fragmentation and membrane integrity values were moderately correlated (r = 0.304, p < 0.05). Fertilisation rates and proportions of eyed eggs showed substantial individual variation and were correlated (r = 0.497, p < 0.05). However, large differences between proportion of eyed eggs and fertilisation rate, median 52% and 81.6% respectively, highlight that the main loss occurred due to embryo mortality rather than failed fertilisation. No correlation was found between either DNA fragmentation or membrane integrity and the resulting reproductive outcome (fertilisation rate and eyed eggs) of the individual Arctic charr sires. Overall, our study identified very high levels of DNA fragmentation, which could influence the fertility of the broodstock in question and thereby be a mitigating mechanism involved in the low reproductive success most often observed in farmed Arctic charr. Further exploration of this relationship would be needed, though.

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.

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 h. 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 production, membrane integrity, and chromatin integrity) were analysed at 0, 24 and 48 h using computer-assisted sperm analysis and flow cytometry. Sperm quality was better in SLC than in CON at all time points, especially chromatin integrity and membrane integrity (P<0.0001 for both), and some categories of reactive oxygen species production (e.g. proportion of live hydrogen peroxide negative spermatozoa, P<0.0001), but the degree of improvement varied among stallions and type of ROS (P<0.05 - P<0.0001). Total and progressive motility were also better in SLC samples than in CON at 24 h and 48 h (P<0.0001), 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.