Sperm quality parameters, fertilizing potential, metabolites, and DNA methylation in cold-stored and cryopreserved milt from Atlantic salmon (Salmo salar L.).

Cold storage and freezing/thawing of milt may affect sperm functionality and the subsequent fertilization ability of milt. This study aimed to investigate sperm quality parameters and fertilization potential of Atlantic salmon milt, stored cold and subsequently cryopreserved, using different storage conditions. The objective was also to assess if analysis of milt metabolites and sperm DNA methylation signatures could be applicable to further elucidate sperm quality and fertilization following preservation. Milt samples were collected from eight mature Atlantic salmon males and stored for 4 days at 2°C and 8°C. Samples were taken on day one of storage at 2°C and on day four of storage at 2°C and 8°C. Storage for 4 days at 8°C is expected to be detrimental to sperm quality, and was included to create contrasts. Correspondingly, aliquots of cold-stored milt were prepared for cryopreservation, resulting in a total of six experimental conditions. Samples from all six experimental conditions were used in fertilization trials and analyzed for sperm viability, motility, ATP content, DNA fragmentation index, and High DNA stainability. In addition, milt samples from four of the males were analyzed for targeted metabolites and DNA methylation signatures by reduced representation bisulfite sequencing. The fertilization trials were performed using sperm:egg ratios of 75 × 103 and 500 × 103, respectively. Storage duration, temperature, and cryopreservation of cold-stored milt influenced several sperm quality parameters, metabolites, and DNA methylation signatures. The total motility, progressive motility, ATP, and velocity parameters were the sperm parameters with the strongest correlation to fertilization rates (p < 0.01). Several metabolites were correlated with fertility rates in both cold-stored and cryopreserved samples (p < 0.05). The fertilizing capacity of cold-stored milt was significantly reduced after 4 days of storage at 8°C, while corresponding cryopreserved milt showed reduced fertilization at both storage temperatures (2°C and 8°C) (p < 0.05). The results indicate that cryopreservation of milt stored for 1 day does not compromise either fertilization ability or DNA methylation signatures.

Ovarian characteristics and in vitro nuclear and cytoplasmic oocyte maturation in Duroc and Landrace pigs.

Differences in total number of piglets born per litter are observed between the Norwegian Duroc (ND) sire and Norwegian Landrace (NL) dam line. The aim of this study was to evaluate ovarian characteristics, and in vitro nuclear and cytoplasmic oocyte maturation in both breeds. One day after weaning, follicular phase ovaries were collected. Ovary length and weight were measured and the number of follicles (< 3 mm and 3-8 mm) was counted. Cumulus-oocyte complexes (COCs) were collected and matured for 48 hr. To assess cumulus expansion, COC area was analysed at 0 and 20 hr. Nuclear maturation and cortical granule (CG) distribution were analysed at 20 and 48 hr, and total glutathione (GSH) was measured at 48 hr to further elucidate cytoplasmic maturation. In first parity sows, a smaller ovary length and fewer 3 to 8 mm follicles were observed in ND compared to NL. For all sows, ND COCs covered a significantly smaller area at 0 hr, but a higher cumulus expansion ratio was observed at 20 hr compared to NL (364 ± 46% versus. 278 ± 27%, p < 0.001). At 20 hr, more ND oocytes exhibited advanced stages of nuclear maturation, while more NL oocytes showed advanced stages of CG distribution. Nuclear maturation to MII stage at 48 hr did not differ between ND and NL oocytes (90.1% and 87.7%, respectively). Moreover, no significant differences were observed for GSH content or CG distribution after maturation. In conclusion, differences with regard to ovarian characteristics as well as to cumulus expansion, and nuclear and cytoplasmic oocyte maturation at 20 hr were observed between the breeds. Further studies are required to determine if this subsequently affects in vitro fertilization and embryo development.

Viability, motility, ATP content and fertilizing potential of sperm from Atlantic salmon (Salmo salar L.) in milt stored before cryopreservation.

Artificial fertilization is increasingly used in aquaculture, mostly applying short-term cold stored milt. Large scale cryopreservation of milt could be valuable for increased flexibility and acceleration of breeding progress. The aim of this study was to assess viability, motility and ATP content of sperm from Atlantic salmon as a function of storage time, before and after cryopreservation. The objective was also to investigate whether in vitro parameters were associated with sperm fertilizing ability after cryopreservation. Milt from six mature Atlantic salmon males were collected twice, one week apart. The milt was stored undiluted at 5 °C in cell culture flasks for six days. Samples were taken on days 1, 3 and 6 of storage for cryopreservation. In total, 36 batches were diluted to a standardized sperm concentration of 2 × 109 spermatozoa/mL, filled into 0.5 mL French medium straws and cryopreserved. In vitro analyses were assessed on the same sample for the 72 combinations of male, collection week, days of storage and cold stored or frozen-thawed. Fertilization trials with cryopreserved milt were carried out for all 36 batches in triplicate for each combination of male, collection week, storage time and sperm:egg ratios of either 2 or 4 × 106 sperm per egg, respectively, totally 218 experimental units, including two egg controls. There was a significant influence of storage and collection week on sperm quality parameters, both cold stored and cryopreserved, and cryopreservation had a significant effect on all tested sperm quality parameters. High correlations for cold stored vs cryopreserved samples was demonstrated for ATP content (p < 0.00001), motility and velocity parameters (p < 0.001), but not for viability, straightness and linearity. The overall percentage of fertilization achieved was 73.9 ± 1.7%. Sperm collected in week 2 showed significantly lower fertility when cryopreserved after six days of storage than after 1 or 3 days for sperm to egg ratios of 2 × 106 (p < 0.005), while there was no such effect for milt collected in week 1. Several post-thaw sperm parameters were correlated to fertilization rates, while curvilinear velocity best explained variations in fertilization by modelling. Our results suggest that cryopreservation of Atlantic salmon milt should be performed soon after milt collection to maximize the cryopreserved sperm quality. Fertilization results seems not to be compromised by storage for three days before cryopreservation.

Sperm DNA integrity in Landrace and Duroc boar semen and its relationship to litter size.

The sperm chromatin structure assay is a method for assessment of sperm DNA fragmentation, a parameter reported to be negatively related to field fertility in several mammal species. This method calculates a DNA fragmentation index (DFI) whose high values indicate abnormal chromatin structure. In this study, running from March 2010 until June 2017, the aim was to assess sperm DFI in stored liquid extended semen from two different pig breeds, Norwegian Landrace (NL; n = 693) and Norwegian Duroc (ND; n = 655), and to evaluate the influence on total number of piglets born (TNB). There was a significantly higher median DFI (p < 0.0001) in ejaculates from the 478 ND boars compared to the 452 NL boars. Data from 19,496 NL litters and 3,877 ND litters of the same boars were retrieved. For either breed, sow herd (p < 0.0001), parity (p < 0.05) and DFI (p < 0.05) showed significant effects on TNB. The DFI was negatively correlated to TNB in both breeds. The boars with the 5% lowest TNB had a least square means DFI of 3.05% and 2.24% in NL and ND, respectively, compared to 1.67% and 1.23% for the boars with the 5% highest TNB (p < 0.01). The DFI and the motility of the same semen samples were negatively correlated (p < 0.0001), and the high and low TNB groups showed significant differences in motility. However, this difference could not be used for practical prediction of TNB group (92.1% vs. 89.7%; p = 0.0038 and 92.3% vs. 89.5%; p = 0.018; NL and ND, respectively). In conclusion, our results indicate that sperm DNA integrity in semen with good motility and morphology may be an additional prediction parameter for fertility in pigs.

Stress Resilience of Spermatozoa and Blood Mononuclear Cells without Prion Protein.

The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrPC (PRNPTer/Ter) compared with cells from normal (PRNP+/+) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS), revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.