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.publishedVersio

Sperm DNA Hypomethylation Proximal to Reproduction Pathway Genes in Maturing Elite Norwegian Red Bulls

Genomic selection in modern farming demands sufficient semen production in young bulls. Factors affecting semen quality and production capacity in young bulls are not well understood; DNA methylation, a complicated phenomenon in sperm cells, is one such factors. In this study, fresh and frozen-thawed semen samples from the same Norwegian Red (NR) bulls at both 14 and 17 months of age were examined for sperm chromatin integrity parameters, ATP content, viability, and motility. Furthermore, reduced representation bisulfite libraries constructed according to two protocols, the Ovation R RRBS Methyl-Seq System (Ovation method) and a previously optimized gel-free method and were sequenced to study the sperm DNA methylome in frozen-thawed semen samples. Sperm quality analyses indicated that sperm concentration, total motility and progressivity in fresh semen from 17 months old NR bulls were significantly higher compared to individuals at 14 months of age. The percentage of DNA fragmented sperm cells significantly decreased in both fresh and frozen-thawed semen samples in bulls with increasing age. Libraries from the Ovation method exhibited a greater percentage of read loss and shorter read size following trimming. Downstream analyses for reads obtained from the gel-free method revealed similar global sperm DNA methylation but differentially methylated regions (DMRs) between 14- and 17 months old NR bulls. The majority of identified DMRs were hypomethylated in 14 months old bulls. Most of the identified DMRs (69%) exhibited a less than 10% methylation difference while only 1.5% of DMRs exceeded a 25% methylation difference. Pathway analysis showed that genes annotated with DMRs having low methylation differences (less than 10%) and DMRs having between 10 and 25% methylation differences, could be associated with important hormonal signaling and sperm function relevant pathways, respectively. The current research shows that RRBS in parallel with routine sperm quality analyses could be informative in reproductive capacity of young NR bulls. Although global sperm DNA methylation levels in 14 and 17 months old NR bulls were similar, regions with low and varying levels of DNA methylation differences can be identified and linked with important sperm function and hormonal pathways.publishedVersio

Semen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bulls

Genomic selection in cattle breeding has gradually allowed younger bulls to be recruited for semen production. In this study, sperm quality parameters, seminal plasma and sperm metabolites, semen production capacity and fertility in young Norwegian Red bulls were analysed. For in vitro analyses of sperm quality and metabolites, ejaculates were collected from the same 25 bulls at both 14 and 17 months of age. Semen production and fertility data were collected for all Norwegian Red bulls in production from December 2017 throughout 2019. Bull fertility was measured as 56 days non-return rate (NR56), for both age groups.acceptedVersionpublishedVersio

Semen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bulls

Genomic selection in cattle breeding has gradually allowed younger bulls to be recruited for semen production. In this study, sperm quality parameters, seminal plasma and sperm metabolites, semen production capacity and fertility in young Norwegian Red bulls were analysed. For in vitro analyses of sperm quality and metabolites, ejaculates were collected from the same 25 bulls at both 14 and 17 months of age. Semen production and fertility data were collected for all Norwegian Red bulls in production from December 2017 throughout 2019. Bull fertility was measured as 56 days non-return rate (NR56), for both age groups. In both fresh and frozen-thawed semen samples, the proportion of hyperactive spermatozoa, average path velocity, curvilinear velocity and amplitude of lateral head displacement were higher in samples collected at 17 months of age compared to 14 months (PSemen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bullsacceptedVersio

Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs

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Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs

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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.publishedVersio

Use of immobilized cryopreserved bovine semen in a blind artificial insemination trial

AbstractTo make timing of artificial insemination (AI) relative to ovulation less critical, methods for prolonging shelf life of spermatozoa in vivo after AI have been attempted to be developed. Encapsulation of sperm cells is a documented technology, and recently, a technology in which sperm cells are embedded in alginate gel has been introduced and commercialized. In this study, standard processed semen with the Biladyl extender (control) was compared with semen processed by sperm immobilization technology developed by SpermVital AS in a blind field trial. Moreover, in vitro acrosome and plasma membrane integrity was assessed and compared with AI fertility data for possible correlation. Semen from 16 Norwegian Red young bulls with unknown fertility was collected and processed after splitting the semen in two aliquots. These aliquots were processed with the standard Biladyl extender or the SpermVital extender to a final number of 12 × 106 and 25 × 106 spermatozoa/dose, respectively. In total, 2000 semen doses were produced from each bull, divided equally by treatment. Artificial insemination doses were set up to design a blinded AI regime; 5 + 5 straws from each extender within ejaculates in ten-straw goblets were distributed to AI technicians and veterinarians all over Norway. Outcomes of the inseminations were measured as 56-day nonreturn rate (NRR). Postthaw sperm quality was assessed by flow cytometry using propidium iodide and Alexa 488–conjugated peanut agglutinin to assess the proportion of plasma membrane and acrosome-intact sperm cells, respectively. In total, data from 14,125 first inseminations performed over a 12-month period, 7081 with Biladyl and 7044 with SpermVital semen, were used in the statistical analyses. There was no significant difference in 56-day NRR for the two semen categories, overall NRR being 72.5% and 72.7% for Biladyl and SpermVital, respectively. The flow cytometric results revealed a significant higher level of acrosome-intact live spermatozoa in Biladyl-processed semen compared to SpermVital semen. The results indicate that the level of acrosome-intact live spermatozoa in the AI dose did not affect the 56-day NRR for the two semen processing methods. In conclusion, this study has showed that immobilized spermatozoa provide equal fertility results as standard processed semen when AI is performed in a blinded field trial, although the immobilization procedure caused increased sperm damage evaluated in vitro compared to standard semen processing procedure

Effect of two 'progressively motile sperm-oocyte' ratios on porcine in vitro fertilization and embryo development

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

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