Anette Kristine Krogenæs

The conservation of local poultry genetic resources requires complementary ex situ strategies, particularly when live populations are maintained at a single location. Previous gene bank insemination trials with frozen–thawed semen from Norwegian conserved lines showed acceptable subjective post-thaw motility but unexpectedly low fertilization success, highlighting the need for objective quality-control assessment. The aim of this study was to establish an objective cryobank quality-control baseline for frozen–thawed semen from two Norwegian conserved rooster lines. Specifically, the study evaluated whether routinely applied objective semen-quality assessment methods, including computer-assisted semen analysis (CASA) and flow-cytometric viability assessment, could detect differences between rooster lines and post-thaw handling conditions. Cryopreserved semen doses collected in 2018 and stored in the Norwegian poultry gene bank were thawed and evaluated from 24 roosters representing the Jærhøns and NorBrid8 lines. The samples were originally collected as part of the routine cryopreservation activities of the Norwegian poultry gene bank and therefore did not require specific approval from the Norwegian Food Safety Authority (Mattilsynet). Post-thaw semen quality was assessed using CASA and flow-cytometric viability analysis. The effects of line and short-term refrigerated holding were evaluated using mixed-effects models, supported by minimum detectable effect calculations, correlation analysis, and principal component analysis. Short-term post-thaw refrigerated holding reduced several CASA-derived motility and velocity traits, while viability assessment provided complementary information on membrane integrity. Line-associated differences in selected motility traits and substantial rooster-to-rooster variability in all traits were observed. Multivariate analyses identified major dimensions of covariance among sperm-quality traits without implying direct biological mechanisms of fertility failure. CASA and flow-cytometric viability assessment can provide an objective baseline for cryobank quality control of frozen–thawed rooster semen. However, fertility trials and molecular sperm-integrity markers are required to determine the biological causes of the previously observed discrepancy between motility and fertility.

Japanese medaka (Oryzias latipes) has been used as a model organism in different research fields, including reproductive physiology. Sperm motility is the most important marker for male fertility in fish and, thus, reproduction success. However, because of small volume of ejaculate and short motility duration, it is still challenging to manage the sperm collection and analysis in small model fish. In the present study, we aimed to investigate sperm motility and to optimize sperm collection, short-term sperm storage, and cryopreservation in Japanese medaka (Oryzias latipes). Using two different approaches for sperm collection: testes dissection and abdominal massage, different housing conditions and activating the sperm with different activation solutions, we investigated immediate sperm motility. In the second part of this study, we used different osmolalities of immobilization solution, Hank's Balanced Salt Solution (HBSS) for sperm storage at 0, 2 and 3 h after sperm collection. Finally, the sperm were cryopreserved using methanol as cryoprotectant and HBSS as extender at two different osmolalities, and post-thaw sperm motility was investigated. The highest post-activating sperm motility was achieved in the groups activated by the extender at 300 mOsm/kg. The quality of sperm remained unaffected by co-housing with females or with males only. Furthermore, Hanks’ Balanced Salt Solution (HBSS) with an osmolality of 600 mOsm/kg demonstrated its efficacy as a suitable extender for sperm storage, preserving motility and progressivity for 3 h. The highest post-thaw motility was around 35%. There were no significant differences between post-thaw motility in different groups. We also found that post-thaw incubation on ice can maintain the motility of the sperm for up to one hour after thawing.

Introduction: Cervical artificial insemination (AI) with frozen-thawed semen in sheep has yielded unacceptably low pregnancy rates. The exception is in Norway where vaginal AI yields non-return rates in excess of 60%, which has been attributed to the ewe breed used. Objectives and methods: This study aimed to characterise, for the first time, the ovine follicular phase cervical mucus metabolome, with a focus on the amino acid profile. Cervical mucus was collected from four European ewe breeds with known differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk (low fertility), Belclare (medium fertility), Norwegian White Sheep (NWS) and Fur (both high fertility). Results: A total of 689 metabolites were identified in the cervical mucus of all the four ewe breeds. Of these, 458 metabolites were altered by ewe breed, which had the greatest effect in the dataset (P < 0.05). We detected 194 metabolites involved in the amino acid pathway, of which 133, 56 and 63 were affected by ewe breed, type of cycle and their interaction, respectively (P < 0.05). N-methylhydantoin and N-carbamoylsarcosine (degradation products of creatinine pathway) exhibited the greatest fold change decrease in the Suffolk breed compared to Fur and NWS (P < 0.001). Oxidized metabolites were also decreased in Suffolk compared to high fertility breeds (P < 0.05). In contrast, other metabolites such as 3-indoxyl-sulfate, putrescine, cadaverine were significantly increased in Suffolk at the synchronised cycle. Conclusion: The suboptimal amino acid profile in the cervical mucus of the low fertility Suffolk breed may have negative consequences for sperm transport.

Subclinical endometritis (SCE) is highly prevalent in dairy cows, causing negative effects on reproductive outcomes and the producer economy. Genetic selection for animals with better resilience against uterine disease should be prioritized due to both sustainability and animal welfare. Therefore, the aim of the present study was to estimate the heritability of SCE in the Norwegian Red (NR) population. Moreover, future perspectives of the condition as a fertility phenotype for breeding are discussed. A total of 1,642 NR cows were sampled for SCE at the time of artificial insemination, using cytotape. The percentage of polymorphonuclear cells (PMN) in each sample was established by cytology, through the counting of 300 PMN and epithelial cells. The mean percentage of PMN was 5%. Different trait definitions were examined, and SCE was defined as binary traits, based on the following cut-off levels of PMN: Cyto0 = PMN >0, Cyto3 = PMN >3%, Cyto5 = PMN >5%, Cyto10 = PMN >10%, and Cyto20 = PMN >20%. The mean ranged from 0.07 (Cyto20) to 0.59 (Cyto0). We also analyzed PMN as a continuous variable using percent PMN. Information on the animals and herds was obtained from the Norwegian Dairy Herd Recording System. The pedigree of cows with data included a total of 24,066 animals. A linear animal model was used to estimate the heritability. The only trait definition that had an estimated genetic variance larger than the standard error was Cyto5, with an estimated heritability of 0.04. For all other definitions, the genetic variance was not significantly different from zero. A cut-off level of 5% PMN has been established as a general threshold for the definition of SCE in earlier literature. The standard errors of the estimated variance components were relatively large, and results should be interpreted with caution. However, the current study indicates that SCE is heritable at a similar level to that of clinical endometritis and metritis, and has potential as a future fertility phenotype to be used for breeding purposes. A more feasible method to diagnose SCE is needed to establish larger data sets.

During the last decade, paternal effects on embryo development have been found to have greater importance than previously believed. In domestic cattle, embryo mortality is an issue of concern, causing huge economical losses for the dairy cattle industry. In attempts to reveal the paternal influence on embryo death, recent approaches have used transcriptome profiling of the embryo to find genes and pathways affected by different phenotypes in the bull. For practical and economic reasons, most such studies have used in vitro produced embryos. The aim of the present study was to investigate the differences in the global transcriptome of in vivo produced embryos, derived from sires with either high or low field fertility measured as the non-return rate (NRR) on day 56 after first AI of the inseminated cows. Superovulated heifers (n = 14) in the age span of 12–15 months were artificially inseminated with semen from either high fertility (n = 6) or low fertility (n = 6) bulls. On day seven after insemination, embryos were retrieved through uterine flushing. Embryos with first grade quality and IETS stage 5 (early blastocyst), 6 (blastocyst) or 7 (expanded blastocyst) were selected for further processing. In total, RNA extracted from 24 embryos was sequenced using Illumina sequencing, followed by differential expression analysis and gene set enrichment analysis. We found 62 genes differentially expressed between the two groups (adj.p-value<0.05), of which several genes and their linked pathways could explain the different developmental capacity. Transcripts highly expressed in the embryos from low fertility bulls were related to sterol metabolism and terpenoid backbone synthesis, while transcripts highly expressed in the high fertility embryos were linked to anti-apoptosis and the regulation of cytokine signaling. The leukocyte transendothelial migration and insulin signaling pathways were associated with enrichments in both groups. We also found some highly expressed transcripts in both groups which can be considered as new candidates in the regulation of embryo development. The present study is an important step in defining the paternal influence in embryonic development. Our results suggest that the sire’s genetic contribution affects several important processes linked to pre-and peri implantation regulation in the developing embryo.

The present study aimed to assess the occurrence of cytological endometritis (CYTO), a nonsymptomatic inflammation of the endometrium, at first artificial insemination (AI) postpartum in Norwegian Red cows. Further, risk factors for CYTO manifestation and its effect on reproductive success and late embryo loss were evaluated. In total 1,648 cows located in 116 herds were included in the study. On mainly spontaneous estrus, endometrial cytology samples were collected using a cytotape technique, and a total of 300 representative epithelial cells and polymorphonuclear neutrophils (PMN) were counted at 400× magnification. Vaginal mucus obtained by Metricheck (Simcro) and body condition score were recorded. Milk samples for progesterone analysis were collected at AI and 21 d later. Pregnancy was diagnosed by rectal palpation or analysis of pregnancy-associated glycoproteins. Based on the constructions of a receiver operator characteristics curve, the cut-off level for PMN defined as CYTO was set to 3.0%, representing the level at which the PMN occurrence affected pregnancy outcome, with the highest summation of sensitivity (32.4%) and specificity (74.9%). Three logistic models with herd included as random factor were constructed. The outcome for the first model was the likelihood for CYTO based on the endometrial samples, in the second model pregnancy to first AI, and in the third model embryo loss. The proportion of CYTO was 28.0% (461/1,648). The average interval in days to first AI was 71.7 d (standard error ± 0.7) and the overall pregnancy incidence to first AI was 59.8% (866/1,449). The likelihood for CYTO at first AI was associated with AI personnel, calving to first AI interval, vaginal mucus characteristics, amount of red blood cells in sample, season, and barn type. Pregnancy to first AI was lower in CYTO-positive cows (odds ratio = 1.51, confidence interval = 1.17-1.94). Other factors affecting pregnancy to first AI were AI personnel, test day milk yield, barn type, and obstetrical conditions or fertility treatments before first AI. The proportion of late embryo loss and abortion was 8.6% (82/948) and 2.8% (24/866), respectively. Late embryo loss was associated with treatment against fertility disorders before first AI, but not associated with CYTO. Overall, our results suggest that even if Norwegian Red cows show a fairly high prevalence of CYTO in the endometrium at first AI, it does not seem to have a major effect on the reproductive performance. The Norwegian Red breeding program has emphasized fertility and health for decades, and a genetically advantageous uterine immunology might be one of the preserved mechanisms.

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.

The most prevalent Fusarium mycotoxin in grains is deoxynivalenol (DON). Contamination of swine feed with DON can result in reduced consumption and poor growth performance. Gestating and lactating sows need sufficient feed intake for fetus development during late gestation and milk production and body maintenance during lactation. Therefore, there is considerable concern in modern piglet production about the effects of DON contamination in sow feed. Most previous studies in sows have been done under experimental conditions, with DON levels ≥2.8 mg/kg feed. The aim of the current field trial was to investigate the effects of feeding grains that are naturally contaminated with more realistic levels of DON on sows during late gestation and lactation. In a commercial, high-yield specific pathogen-free piglet production unit, 45 Norwegian Landrace × Yorkshire sows were fed three diets from 93 ± 1 days of gestation until weaning of the piglets, and average daily feed intake (ADFI), body weight (BW), production and reproduction performance, as well as sow blood parameters were recorded. Diets were made from naturally contaminated oats, with three concentration levels: 1) control (DON < 0.2 mg/kg), 2) DON level 1 (1.4 mg DON/kg), and 3) DON level 2 (1.7 mg DON/kg). Sows that were fed DON level 1 and 2 diets showed a 4–10% reduction in feed consumption during lactation, compared with sows in the control group. However, the DON-contaminated diets did not significantly affect sow BW or backfat thickness. Similarly, there were neither effects on production or reproduction performance, nor on blood parameters in the sows. The effects on skin temperature were variable. Naturally contaminated diets with realistic, moderately increased DON levels, fed during late gestation and lactation in a modern high-yield piglet production farm, had limited effects on sow health and production.