The microbiota of uterine biopsies, cytobrush and vaginal swabs at artificial insemination in Norwegian red cows.

The individual resistance or tolerance against uterine disease in dairy cattle might be related to variations in the uterine tract microbiota. The uterine tract microbiota in dairy cattle is a field of increasing interest. However, its specific taxonomy and functional aspects is under-explored, and information about the microbiota in the endometrium at artificial insemination (AI) is still missing. Although uterine bacteria are likely to be introduced via the vaginal route, it has also been suggested that pathogens can be transferred to the uterus via a hematogenous route. Thus, the microbiota in different layers of the uterine wall may differ. Norwegian Red (NR) is a high fertility breed that also has a high prevalence of subclinical endometritis (SCE), an inflammation of the uterus that has a negative effect on dairy cattle fertility. However, in this breed the negative effect is only moderate, raising the question of whether this may be due to a favorable microbiota. In the present study we investigated the endometrial microbiota in NR at AI by biopsy and cytobrush samples, and comparing this to the vaginal microflora. The second objective was to describe potential differences at both distinct depths of the endometrium, in healthy vs SCE positive NR cows. We sampled 24 lactating and clinically healthy Norwegian red cows in their second heat or more after calving, presented for first AI. First, we obtained a vaginal swab and a cytobrush sample, in addition to a cytotape to investigate the animal's uterine health status with respect to SCE. Secondly, we acquired a biopsy sample from the uterine endometrium. Bacterial DNA from the 16S rRNA gene was extracted and sequenced with Illumina sequencing of the V3-V4 region. Alpha and beta diversity and taxonomic composition was investigated. Our results showed that the microbiota of endometrial biopsies was qualitatively different and more even than that of cytobrush and vaginal swab samples. The cytobrush samples and the vaginal swabs shared a similar taxonomic composition, suggesting that vaginal swabs may suffice to sample the surface-layer uterine microbiota at estrus. The current study gave a description of the microbiota in the healthy and SCE positive NR cows at AI. Our results are valuable as we continue to explore the mechanisms for high fertility in NR, and possible further improvements.

Gene Expression in Embryos From Norwegian Red Bulls With High or Low Non Return Rate: An RNA-Seq Study of in vivo-Produced Single Embryos.

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.

Intra- and Inter-Observer Reliability of Qualitative Behaviour Assessments of Housed Sheep in Norway.

This study tested the reliability of a Qualitative Behavioural Assessment (QBA) protocol developed for the Norwegian Sheep House (FåreBygg) project. The aim was to verify whether QBA scores were consistent between different observers, i.e., inter-observer reliability, and between scorings of the same observers on different time points, i.e., intra-observer reliability. Six trained observers, including two veterinary students, two animal welfare inspectors and two sheep farmers observed sheep in 16 videos, and independently scored 14 pre-defined behavioural descriptors on visual analogue scales (VAS). The procedure was repeated one week after the first scoring session. QBA scores were analysed using Principal Component Analysis. Inter- and intra-observer agreement was assessed using Kendall's coefficient of concordance (W). Principal component 1 (PC 1) and 2 (PC 2) combined explained >60% of the total variation in the QBA scores in both scoring sessions. PC 1 (44.5% in sessions 1 and 2) ranged from the positive descriptors calm, content, relaxed and friendly to the negative descriptors uneasy, vigilant and fearful, and was therefore labelled mood. PC 2 (18% in session 1, 16.6% in session 2) ranged from bright to dejected and apathetic, and was therefore labelled arousal. Kendall's coefficient of concordance of PC 1 for all observers was high in the two scoring sessions (W = 0.87 and 0.85, respectively), indicating good inter-observer reliability. For PC 2, the agreement for all observers was moderate in both video sessions (W = 0.45 and 0.65). The intra-observer agreement was very high for all observers for PC 1 (W > 0.9) except for one, where the agreement was considered to be high (W = 0.89). For PC 2, Kendall's coefficient was very high for the veterinary students and interpreted as moderate for the two farmers and welfare inspectors. This study indicates that the QBA approach and the terms included in the Fårebygg protocol were reliable for assessing video recordings of sheep behaviour when applied by trained observers, regardless of whether they were a veterinary student, animal welfare inspector or sheep farmer. Further work is needed to examine the reliability of the QBA protocol when tested on-farms for sheep managed under Norwegian housing systems.