| 1. |
- Bezerra Lima Verde, Isabel, et al.
(författare)
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Effect of freezing bull semen in two non-egg yolk extenders on post-thaw sperm quality
- 2018
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Ingår i: Reproduction in Domestic Animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 53, s. 127-136
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Tidskriftsartikel (refereegranskat)abstract
- Traditionally, extenders for bull semen included egg yolk or milk, but recently there has been a move to avoid material of animal origin. The aim of this study was to evaluate the effects of two commercial extenders (based on soya lecithin and liposomes) on bull sperm quality after cryopreservation. Post-thaw sperm quality was evaluated by computer-assisted sperm analysis and flow cytometric assessment of membrane integrity, chromatin integrity, mitochondrial membrane potential, production of reactive oxygen species and tyrosine phosphorylation. Furthermore, an artificial insemination (AI) trial was conducted, and 56-day non-return rates were evaluated. Semen frozen in the liposome-based extender showed similar membrane integrity and higher mitochondrial membrane potential compared to those in the soya lecithin-based extender. Chromatin integrity and production of live H2O2+ reactive oxygen species were similar in both extenders. Less superoxide was produced in the samples extended with liposome-based extender, with or without menadione stimulation. Chromatin integrity and tyrosine phosphorylation were not affected by either type of extender. No differences in 56-day non-return rate between extenders containing soya lecithin and liposomes were observed in the AI trial (66%+/- 0.8 and 65%+/- 0.8, respectively). In conclusion, the sperm quality of bull semen frozen in the two extenders that do not contain material of animal origin was similar, although the semen frozen in the liposome-based extender had higher mitochondrial membrane potential. Either extender could be used in situations where extenders containing material of animal origin are to be avoided.
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| 2. |
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| 3. |
- Al-Kass, Ziyad, et al.
(författare)
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Bacteria detected in the genital tract, semen or pre-ejaculatory fluid of Swedish stallions from 2007 to 2017
- 2019
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Ingår i: Acta Veterinaria Scandinavica. - : Springer Science and Business Media LLC. - 0044-605X .- 1751-0147. ; 61
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Tidskriftsartikel (refereegranskat)abstract
- Background: Although artificial insemination (AI) was developed as a means of controlling disease transmission, pathogens can still be transmitted to females in semen used for AI. In addition, bacteria can cause deterioration in sperm quality during storage. Semen becomes contaminated by the male's normal bacterial flora as it passes out of the reproductive tract but potential pathogens may also contaminate the semen. Therefore, semen samples from stallions to be used for AI are tested before the breeding season to minimize transmission of pathogens to inseminated mares. In Sweden, semen samples are tested at the National Veterinary Institute, Uppsala (SVA). For the present study, a retrospective analysis was made of potentially pathogenic bacteria isolated from samples submitted to the SVA from 2007 to 2017.Results: In our study, Taylorella equigenitalis was found infrequently (53 out of 25,512 samples), representing 11 out of 2308 stallions. If T. equigenitalis was detected, the stallions were treated with antibiotics and re-tested later in the same year. Klebsiella pneumoniae and beta haemolytic streptococci were the most commonly found potential pathogens, whereas Pseudomonas aeruginosa was also isolated occasionally. There were considerable differences in the number of species isolated each year.Conclusions: Potential pathogens were identified in relatively few of the samples submitted to SVA during this period, with T. equigenitalis not being identified since 2015. Of the other potential pathogens, K. pneumoniae and beta haemolytic streptococci were the most common. The information is relevant for determining guidelines on the testing and treatment of stallions before breeding.
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| 4. |
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| 5. |
- Al-Kass, Ziyad
(författare)
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Characterization, quantification and removal of potential pathogens from stallion semen
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In Sweden, equine artificial insemination is most frequently carried out with liquid semen rather than frozen semen. Many factors affect sperm quality during semen storage, including the presence of bacteria and addition of antibiotics. Use of Single Layer Centrifugation (SLC) or Modified SLC (MSLC) has been shown to improve sperm quality, resulting in an increase in the time for which the sperm sample retains its function during storage. These techniques can also separate spermatozoa from bacteria. The purpose of this thesis was to investigate the role of MSLC, antibiotics and bacteria on sperm quality and storage time. This thesis comprised 4 studies: Study I was a retrospective study of potential pathogenic bacteria isolated from Swedish stallion semen during the period 2007 to 2017. Study II was to identify bacteria in semen by conventional laboratory culture methods. Study III was to isolate pathogenic and nonpathogenic bacteria in stallion semen using metagenomic analysis. Study IV was to investigate the effect of MSLC and the effect of presence or absence of antibiotics in the extender on sperm quality. Our results showed that potentially pathogenic bacteria such as Taylorella equigenitalis, Klebsiella pneumoniae, beta haemolytic streptococci and Pseudomonas aeruginosa appeared infrequently in Sweden. However, many nonpathogenic bacteria were found. Metagenomic analysis enabled more bacteria to be identified than other methods. The bacterial genera identified were different between studies, animals and ejaculates, even from animals kept on the same stud. Corynebacterium spp. were the most frequently found non-pathogenic bacteria identified in all our studies. The bacterial population was decreased using MSLC, sperm quality was improved and the shelf-life of the sperm samples was increased. The presence of antibiotics in the extender did not affect sperm viability. The bacterial population was greater in samples without antibiotics than in samples with antibiotics; bacteria appeared in all samples, even those with extender containing antibiotics. There was no evidence that bacteria isolated from a stallion on one stud should also be isolated from other individuals on the same premises. More work is needed to investigate the effects of particular bacterial genera on sperm quality. In addition, it would be interesting to investigate the shape and size of bacteria in relation to spermatozoa and the proportions of different bacteria removed using MSLC. Further modifications to the SLC technique might enable the removal of more bacteria.
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| 6. |
- Al-Kass, Ziyad, et al.
(författare)
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Deciphering sperm chromatin properties to predict stallion sperm fertility
- 2023
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Ingår i: Animal Reproduction Science. - : Elsevier BV. - 0378-4320 .- 1873-2232. ; 250
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
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| 8. |
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| 9. |
- Al-Kass, Ziyad, et al.
(författare)
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Effect of presence or absence of antibiotics and use of modified single layer centrifugation on bacteria in pony stallion semen
- 2019
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Ingår i: Reproduction in Domestic Animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 54, s. 342-349
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Tidskriftsartikel (refereegranskat)abstract
- Contents Bacteria contaminate semen during collection and handling. The objective of this study was to identify the bacteria in pony stallion semen, the effects of antibiotics included in commercial semen extenders (lincomycin and spectinomycin) and the effect of modified single layer centrifugation (MSLC), on bacterial load. Ejaculates from six pony stallions, 3 ejaculates per animal, were extended in EquiPlus extender either with or without antibiotics. Aliquots were processed by MSLC to form four treatment groups: control and MSLC with antibiotics (CA and SA, respectively) and control and MSLC without antibiotics (CW and SW, respectively). Bacteriological examinations were carried out within 2hr. Thirty-one species of bacteria were isolated from one or more ejaculates, with Corynebacterium spp. being the most frequently detected. Corynebacterium spp. were present in all ejaculates. The MSLC resulted in a significantly lower total bacterial count than controls (CA vs. SA, p<0.001; CW vs. SW, p<0.0001).
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| 10. |
- Al-Kass, Ziyad, et al.
(författare)
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Freezing stallion semen – what do we need to focus on for the future?
- 2024
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Ingår i: Veterinary Sciences. - 2306-7381. ; 11
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Forskningsöversikt (refereegranskat)abstract
- Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation.
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| 11. |
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| 12. |
- Al-Kass, Ziyad, et al.
(författare)
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Sperm Quality during Storage Is Not Affected by the Presence of Antibiotics in EquiPlus Semen Extender but Is Improved by Single Layer Centrifugation
- 2018
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Ingår i: Antibiotics. - : MDPI AG. - 2079-6382. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Contamination of semen with bacteria arises during semen collection and handling. This bacterial contamination is typically controlled by adding antibiotics to semen extenders but intensive usage of antibiotics can lead to the development of bacterial resistance and may be detrimental to sperm quality. The objective of this study was to determine the effects of antibiotics in a semen extender on sperm quality and to investigate the effects of removal of bacteria by modified Single Layer Centrifugation (MSLC) through a colloid. Semen was collected from six adult pony stallions (three ejaculates per male). Aliquots of extended semen were used for MSLC with Equicoll, resulting in four treatment groups: control and MSLC in extender with antibiotics (CA and SA, respectively); control and MSLC in extender without antibiotics (CW and SW, respectively). Sperm motility, membrane integrity, mitochondrial membrane potential and chromatin integrity were evaluated daily by computer-assisted sperm analysis (CASA) and flow cytometry. There were no differences in sperm quality between CA and CW, or between SA and SW, although progressive motility was negatively correlated to total bacterial counts at 0 h. However, MSLC groups showed higher mean total motility (P < 0.001), progressive motility (P < 0.05), membrane integrity (P < 0.0001) and mitochondrial membrane potential (P < 0.05), as well as better chromatin integrity (P < 0.05), than controls. Sperm quality remained higher in the MSLC groups than controls throughout storage. These results indicate that sperm quality was not adversely affected by the presence of antibiotics but was improved considerably by MSLC.
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| 13. |
- Al-Kass, Ziyad, et al.
(författare)
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Variation among stallions in sperm quality after single layer centrifugation
- 2021
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Ingår i: Reproduction in Domestic Animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 56, s. 848-856
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Tidskriftsartikel (refereegranskat)abstract
- 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 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.
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| 14. |
- Johannisson, Anders, et al.
(författare)
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Simultaneous evaluation of superoxide content and mitochondrial membrane potential in stallion semen samples provides additional information about sperm quality
- 2018
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Ingår i: Animal Reproduction Science. - : Elsevier BV. - 0378-4320 .- 1873-2232. ; 192, s. 290-297
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Tidskriftsartikel (refereegranskat)abstract
- An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24 h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoechst 33258 exclusion were measured and correlated to superoxide production and MMP. Both bivariate correlations between measured parameters as well as multivariate analysis were performed. Measured values in the superoxide/MMP assay did not correlate with other parameters. However, there was a strong negative correlation (r = 0.96 after 0 h, r = 0.95 after 24 h) between membrane integrity and chromatin integrity. Moderate positive correlations were found between motility parameters and membrane integrity, as well as moderate negative correlations between motility parameters and chromatin integrity. The multivariate analysis revealed that membrane integrity, chromatin integrity and motility contributed to the first principal component, while the second was influenced by superoxide/MMP parameters as well as sperm kinematics. Storage of samples for 24 h decreased motility, chromatin integrity and membrane integrity. In conclusion, combined measurement of superoxide and MMP provides additional information not obtained by other assays of sperm quality.
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