In this study, we propose a possible way of obtaining reproductive and respiratory syndrome virus (PRRSV) free offspring from genetically valuable lines of Přeštice black-pied boars com- ming from PRRSV-positive pig breeding herds with the use of artificial insemination (AI). The ejaculates were collected from 4 different lines of boars. Samples of fresh semen were not detected with the virus and 12 sows were inseminated. Blood samples of sows and their offspring were repeatedly tested for the virus but the results were negative. We managed in this way to maintain the endangered population of this breed and obtain PRRSV-free offspring.
In our recent study we demonstrated that the holding of fresh semen in fractionated seminal plasma (SP1, >40 kDa; SP2, <40 kDa), obtained by gel filtration chromatography, significantly improved the sperm quality characteristics following cryopreservation (Wasilewska-Sakowska et al. 2019). In this study we investigated the effect of post-thaw (PT) supplementation of fractionated SP (SP1 and SP2) on the survival of spermatozoa from boars with good and poor semen freezability, GSF and PSF, respectively. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed distinct differences in the protein profiles of SP1 and SP2 from boars with GSF or PSF regarding the number of protein spots. Sperm motility characteristics and the motion patterns, assessed using the computer-assisted sperm analysis (CASA) system, were markedly higher in PT semen supplemented with SP1 and SP2 from boars with GSF. Post-thaw supplementation of either SP1 or SP2 from boars with GSF significantly improved mitochondrial function, plasma membrane and acrosome integrity, and viability during storage. The findings of this study have confirmed that the presence of protective protein components in varying abundance in either fractionated SP from boars with good freezability ejaculates significantly improved the sperm survival following PT storage.
In this study the quality of total RNA, isolated from fresh spermatozoa, was compared between boars with good and poor semen freezability (GSF and PSF, respectively). Semen from 3 boars with GSF exhibited significantly higher total motility, mitochondrial function, plasma membrane integrity and reduced lipid peroxidation compared with 3 boars with PSF after cryo- preservation. There were variations in the quality of RNA isolated from spermatozoa of boars with GSF and PSF. Boars with GSF exhibited mainly full-length, intact RNA, whereas substantial amounts of degraded RNA were detected in spermatozoa from boars with PSF. Further under- standing of the biological relevance of RNAs in sperm function is critical to improve the freezabil- ity of boar semen.
The aim of this study was to compare computer assisted sperm analysis (CASA) results of frozen thawed bull semen using three different chambers. Sixty bull frozen semen samples were thawed (37°C; 30 sec), extended in PBS (30×106 spermatozoa/mL; 37°C) and incubated (37°C; 2 min). Each semen sample was analyzed by CASA [total motility, progressive (pro)/ non-progressive/rapid/medium/slow movement spermatozoa, VCL, VSL, VAP, ALH, BCF, LIN, STR, WOB and hyperactive spermatozoa] using three different chambers: a Makler® chamber (MC; 10 μm); a Leja 4 chamber slide (LC; 20 μm); and a Glass slide covered with a coverslip (GSC; 10.3 μm). The Makler chamber gave higher values compared to both the LC and GSC for almost all examined parameters. No systematic effect was evident between LC and GSC for VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF. Method agreement between MC and LC was generally moderate, between MC and GSC poor and between LC and GSC moderate to good. In general, narrower limits of agreement were found in samples with lower values. In conclusion, the CASA outcomes could be influenced by the analysis chambers. This finding should be taken into consideration when comparing results from different laboratories.
Electrophoretic methods were used to identify protein complexes formed between ostrich egg yolk lipoprotein fractions (LPFo) with seminal plasma (SP) of fractionated ejaculates, and to investigate the effect of these complexes on boar semen quality after cryopreservation. Chromatographic SP fractions (F1, F2 and F3), with or without LPFo solution, were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Comparative electrophoretic analyses of the SP revealed marked differences in the SDS-PAGE protein profiles among boars. Electrophoretic analyses showed that the interactions of LPFo with SP resulted in the appearance of high-intensity protein bands. Spermatozoa were exposed to SP chromatographic fractions originating from F1, F2 and F3, and the whole SP (wSP) before being frozen. Spermatozoa exposed to F1 and F2 exhibited significantly higher post-thaw motility compared to those treated with either F3 or wSP. In most of the boars the proportions of membrane-intact frozen-thawed spermatozoa differed among the treatments, being significantly lower in the wSP-treated samples. The incidence of frozen-thawed spermatozoa with DNA fragmentation was less prevalent in samples exposed to F3 or the wSP. The results of this study confirmed that the interactions of LPFo with fractionated SP during the cooling period contributed to alterations in the sperm membranes, rendering them less susceptible to temperature-related injury.
The aim of the study was to investigate the effect of soybean lecithin as a substitute for egg yolk in milk and tris based extenders in ram semen cryopreservation. Twenty ejaculates were col- lected from four healthy, mature Wrzosówka rams (2-3 years of age). Each ejaculate was divided into four equal aliquots and diluted with four different extenders: 1) milk extender containing 5% egg yolk, 2) milk extender containing 1.5% soybean lecithin, 3) tris extender containing 20% egg yolk, 4) tris extender containing 1.5% soybean lecithin. Extended semen was loaded into 0.25 ml French straws, cooled and frozen in liquid nitrogen vapor. Total motility, curvilinear velocity, plasma membrane integrity and fertilizing ability of sperm were assessed after thawing. Total mo- tility was lower (p<0.05) in tris-soybean lecithin extender when compared to other extenders. Curvilinear velocity was higher (p<0.05) for spermatozoa cryopreserved in milk-soybean lecithin extender compared to other extenders tested. For the percentage of live sperm no significant difference was observed between extenders. The lambing rate were higher (not statistically signifi- cant) in ewes inseminated with semen doses frozen in milk-soybean lecithin extender (42.9%) than in the tris-egg yolk extender (16.7%). In conclusion, replacing the egg yolk with soybean lecithin was effective in milk but not in tris extender.
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