Associated regions for multiple birth in Brown Swiss and Original Braunvieh cattle on chromosomes 15 and 11.

Twin and multiple births have negative effects on the performance and health of cows and calves. To decipher the genetic architecture of this trait in the two Swiss Brown Swiss cattle populations, we performed various association analyses based on de-regressed breeding values. Genome-wide association analyses were executed using ~600 K imputed SNPs for the maternal multiple birth trait in ~3500 Original Braunvieh and ~7800 Brown Swiss animals. Significantly associated QTL were observed on different chromosomes for both breeds. We have identified on chromosome 11 a QTL that explains ~6% of the total genetic variance of the maternal multiple birth trait in Original Braunvieh. For the Brown Swiss breed, we have discovered a QTL on chromosome 15 that accounts for ~4% of the total genetic variance. For Original Braunvieh, subsequent haplotype analysis revealed a 90-kb window on chromosome 11 at 88 Mb, where a likely regulatory region is located close to the ID2 gene. In Brown Swiss, a 130-kb window at 75 Mb on chromosome 15 was identified. Analysis of whole-genome sequence data using linkage-disequilibrium estimation revealed possible causal variants for the identified QTL. A presumably regulatory variant in the non-coding 5' region of the ID2 gene was strongly associated with the haplotype for Original Braunvieh. In Brown Swiss, an intron variant in PRDM11, one 3' UTR variant in SYT13 and three intergenic variants 5' upstream of SYT13 were identified as candidate variants for the trait multiple birth maternal. In this study, we report for the first time QTL for the trait of multiple births in Original Braunvieh and Brown Swiss cattle. Moreover, our findings are another step towards a better understanding of the complex genetic architecture of this polygenic trait.

An ancient positively selected BMPRIB missense variant increases litter size of Mongolian sheep populations following latitudinal gradient.

New gene mutation origination is a driving force for the evolution of organisms. The effect of FecB mutation in BMPRIB gene on the litter size of sheep has been well known for a long time, each copy of the mutant allele increases litter size by 0.4-0.5. However, the origin and adaptive evolution mechanism of FecB mutation are still unclear. Here we carried on the thorough analysis on evolutionary features of BMPRIB gene and found that 150 species as a whole is under purifying selection while sheep lineage shows evidence of positive selection. The results of allele age estimation revealed that the FecB mutation in Mongolian sheep of China originated in Mongolian Plateau at about 5000 years ago. Due the two shape drops in temperature subsequently, Mongolian sheep migrated from north to south following the northern nomadic people. Accordingly, the FecB mutant allele frequency increased, with the lowest in sheep locating at Mongolian plateau (0.01) and the highest in sheep locating at Yangtze River valley (0.96). In conclusion, the FecB mutation in Mongolian sheep of China originated in Mongolian Plateau at about 5000 years ago, and the differentiated litter size of Mongolian sheep might be the result of adaptation to various environments during the migration following latitudinal gradient. This study may well exemplify selection on an ancient variation triggered by drastic ecological shifts, and is also helpful to analyze the adaptive evolution mechanism of economic traits of domestic animals and identify major genes and molecular markers.

Influence of conceptus presence and preovulatory estradiol exposure on uterine gene transcripts and proteins around maternal recognition of pregnancy in beef cattle.

The uterine environment must provide sufficient endocrine conditions and nutrients for pregnancy maintenance and conceptus survival. The objective of this study was to determine the effects of preovulatory estradiol and conceptus presence on uterine transcripts and uterine luminal fluid (ULF) proteins. Beef cows/heifers were synchronized and artificially inseminated (d 0). Uteri were flushed (d 16); conceptuses and endometrial biopsies were collected. Total cellular RNA was extracted from endometrium for RNA sequencing and RT-PCR validation. There were two independent ULF pools made for each of the following groups: highE2/conceptus, highE2/noconceptus, lowE2/conceptus, and lowE2/noconceptus that were analyzed using the 2D LC-MS/MS based iTRAQ method. There were 64 differentially expressed genes (DEGs) and 77 differentially expressed proteins (DEPs) in common among the highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus groups. In summary, the interaction between preovulatory estradiol and the conceptus induces the expression of genes, proteins, and pathways necessary for pregnancy.

Vaginal mucus in mice: developmental and gene expression features of epithelial mucous cells during pregnancy†.

The vagina is the site of copulation and serves as the birth canal. It also provides protection against external pathogens. In mice, due to the absence of cervical glands, the vaginal epithelium is the main producer of vaginal mucus. The development and differentiation of vaginal epithelium-constituting cells and the molecular characteristics of vaginal mucus have not been thoroughly examined. Here, we characterized vaginal mucous cell development and the expression of mucus-related factors in pregnant mice. The vaginal mucous epithelium layer thickened and became multilayered after Day 12 of pregnancy and secreted increasing amounts of mucus until early postpartum. Using histochemistry and transmission electron microscopy, we found supra-basal mucous cells as probable candidates for precursor cells. In vaginal mucous cells, the expression of TFF1, a stabilizer of mucus, was high, and some members of mucins and antimicrobial peptides (MUC5B and DEFB1) were expressed in a stage-dependent manner. In summary, this study presents the partial characterization of vaginal epithelial mucous cell lineage and expression of genes encoding several peptide substances that may affect vaginal tissue homeostasis and mucosal immunity during pregnancy and parturition.

Pregnancy influences the selection of appropriate reference genes in mouse tissue: Determination of appropriate reference genes for quantitative reverse transcription PCR studies in tissues from the female mouse reproductive axis.

Selecting stably expressed reference genes which are not affected by physiological or pathophysiological conditions is crucial for reliable quantification in gene expression studies. This study examined the expression stability of a panel of twelve reference genes in tissues from the female mouse reproductive axis and the uterus. Gene expression studies were carried out using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). cDNA was synthesised from RNA extracted from hypothalami, pituitaries, ovaries and uteri of female mice at ages representing weaning, puberty and adulthood as well as pregnancy (13 ± 1 days post-coitus) (n = a minimum of 3 at each age and at pregnancy). The reference genes examined included 18 s, Actb, Atp5b, B2m, Canx, Cyc1, Eif4a2, Gapdh, Rpl13a, Sdha, Ubc and Ywhaz. The RT-qPCR raw data were imported into the qBASE+ software to analyse the expression stability using GeNorm. These data were also subsequently analysed using other software packages (Delta CT, Normfinder, BestKeeper). A comprehensive ranking was conducted considering all stability rankings generated from the different software analyses. B2m and Eif4a2 deviated from the acceptable range for amplification efficiency and therefore were excluded from the further analyses. The stability of the reference genes is influenced by the software used for the analysis with BestKeeper providing markedly different results than the other analyses. GeNorm analysis of tissues taken at different ages but not including pregnant animals, indicated that the expression of the reference genes is tissue specific with the most stable genes being: in the hypothalamus, Canx and Actb; in the pituitary, Sdha and Cyc1; in the ovary, 18s, Sdha and Ubc; and in the uterus, Ywhaz, Cyc1, Atp5b, 18s and Rpl13a. The optimal number of reference genes to be used was determined to be 2 in the first three tissues while in the uterus, the V-score generated by the GeNorm analysis was higher than 0.15 suggesting that 3 or more genes should be used for normalisation. Inclusion of tissues from pregnant mice changed the reference genes identified as being the most stable: Ubc and Sdha were the most stable genes in the hypothalamus, pituitary and the ovary. The addition of pregnant tissue had no effect on the stability of the genes in uterus (Ywhaz, Cyc1, Atp5b, 18s and Rpl13a). Identification of these stable reference genes will be of use to those interested in studying female fertility and researchers should be alert to the effects of pregnancy on reference gene stability. This study also signifies the importance of re-examining reference gene stability if the experimental conditions are changed, as shown with the introduction of pregnancy as a new factor in this research.

Constitutive Expression of TERT Enhances ß-Klotho Expression and Improves Age-Related Deterioration in Early Bovine Embryos.

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and ß-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of ß-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts' development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/ß-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.

SPP1 expression in the mouse uterus and placenta: implications for implantation†.

Secreted phosphoprotein 1 (SPP1, also known as osteopontin) binds integrins to mediate cell-cell and cell-extracellular matrix communication to promote cell adhesion, migration, and differentiation. Considerable evidence links SPP1 to pregnancy in several species. Current evidence suggests that SPP1 is involved in implantation and placentation in mice, but in vivo localization of SPP1 and in vivo mechanistic studies to substantiate these roles are incomplete and contradictory. We localized Spp1 mRNA and protein in the endometrium and placenta of mice throughout gestation, and utilized delayed implantation of mouse blastocysts to link SPP1 expression to the implantation chamber. Spp1 mRNA and protein localized to the endometrial luminal (LE), but not glandular epithelia (GE) in interimplantation regions of the uterus throughout gestation. Spp1 mRNA and protein also localized to uterine naturel killer (uNK) cells of the decidua. Within the implantation chamber, Spp1 mRNA localized only to intermittent LE cells, and to the inner cell mass. SPP1 protein localized to intermittent trophoblast cells, and to the parietal endoderm. These results suggest that SPP1: (1) is secreted by the LE at interimplantation sites for closure of the uterine lumen to form the implantation chamber; (2) is secreted by LE adjacent to the attaching trophoblast cells for attachment and invasion of the blastocyst; and (3) is not a component of histotroph secreted from the GE, but is secreted from uNK cells in the decidua to increase angiogenesis within the decidua to augment hemotrophic support of embryonic/fetal development of the conceptus.

Notch1 is crucial for decidualization and maintaining the first pregnancy in the mouse†.

The endometrium undergoes a pregnancy-delivery-repair cycle multiple times during the reproductive lifespan in females. Decidualization is one of the critical events for the success of this essential process. We have previously reported that Notch1 is essential for artificial decidualization in mice. However, in a natural pregnancy, the deletion of Notch1 (PgrCre/+Notch1f/f, or Notch1d/d) only affects female fertility in the first 30 days of a 6-month fertility test, but not the later stages. In the present study, we undertook a closer evaluation at the first pregnancy of these mice to attempt to understand this puzzling phenomenon. We observed a large number of pregnancy losses in Notch1d/d mice in their first pregnancy, which led to the subfertility observed in the first 30 days of the fertility test. We then demonstrated that the initial pregnancy loss is a consequence of impaired decidualization. Furthermore, we identified a group of genes that contribute to Notch1 regulated decidualization in a natural pregnancy. Gene ontogeny analysis showed that these differentially expressed genes in the natural pregnancy are involved in cell-cell and cell-matrix interactions, different from genes that have been previously identified from the artificial decidualization model, which contribute to cell proliferation and apoptosis. In summary, we determined that Notch1 is essential for normal decidualization in the mouse uterus only in the first pregnancy but not in subsequent ones.

Effects of dietary n-3-PUFA supplementation, post-insemination plane of nutrition and pregnancy status on the endometrial transcriptome of beef heifers.

Supplementation of cattle diets with n-3-polyunsaturated fatty acids (PUFA) can improve reproductive efficiency. Conversely, short-term fluctuations in feed supply can impact pregnancy establishment. The objectives of this study were to examine the effects of (1) dietary supplementation with n-3-PUFA and (2) post-insemination plane of nutrition on the endometrial transcriptome. Beef crossbred heifers were offered concentrate based diets fortified with n-3-PUFA (PUFA; n = 32) or not (CONT; n = 28) for 30 days prior to breeding at a synchronised oestrous. Following artificial insemination, heifers were allocated within treatment to either a high or low plane of nutrition. Heifers were maintained on these diets for 16 days following which endometrial tissue was harvested at slaughter for subsequent RNAseq analysis. The influence of pregnancy status on the endomentrial transcriptome, within each dietary treatment group, was also examined. Post-insemination diet affected (P < 0.05) the endometrial transcriptome. Specifically, within n-3-PUFA-supplemented heifers, genes involved in embryonic development and mTOR signalling pathways, important in pregnancy establishment, were identified as differentially expressed. Results indicate that dietary supplementation of cattle diets with n-3-PUFA may have a positive effect on the expression of key fertility-related genes and pathways, during the critical window of maternal recognition of pregnancy, particularly where animals are underfed.

Phenotypic and genetic effects of pregnancy on milk production traits in Holstein-Friesian cattle.

Pregnancy is a prerequisite for the initiation of lactation and for maintaining the milk production cycle. Pregnancy affects milk production and therefore should be accounted for in the genetic evaluation. Furthermore, there might be genetic differences in pregnancy effects on milk composition. The objective of this study was to estimate phenotypic and genetic effects of pregnancy on milk production traits. For this purpose, test-day records and conception dates of 1,359 first-parity Holstein-Friesian cows were analyzed. Significant effects of pregnancy on all milk production traits were detected except somatic cell score (e.g., the cumulative effects of pregnancy on milk yield were -247 kg). The pregnancy effects on milk yield, lactose yield, protein yield, fat yield, and fat content were small during early gestation (<150 d) and substantially increased in late gestation. The effects of pregnancy on milk protein yield were relatively stronger than those on fat yield. The effects of pregnancy on milk production traits differed for DGAT1 genotypes. Milk yield, lactose yield, protein yield, and fat yield of DGAT1 AA cows were more affected by pregnancy than that of DGAT1 KK cows (e.g., the cumulative effects of pregnancy on milk yield were negligible for DGAT1 KK cows and were -443 kg for DGAT1 AA cows). These results suggest that DGAT1 KK cows may be more suitable for shortening or omitting the dry period than DGAT1 AA cows.

Identification and characterization of miRNAs during early pregnancy in domestic sheep.

MicroRNA resources in sheep are limited compared with those in other domesticated mammalian species. By sequencing small RNAs of sheep corpus luteum and endometrium, we have generated the largest amount of miRNA-seq data and compiled the most comprehensive list thus far of miRNAs (n = 599) in sheep. Additionally, we observed a highly conserved maternally imprinted cluster of miRNAs on chromosome 18 homologous to that found on chromosome 14 in human and several other eutherian mammals.

Relative abundance of chemerin mRNA transcript and protein in pituitaries of pigs during the estrous cycle and early pregnancy and associations with LH and FSH secretion during the estrous cycle.

Adipokines such as chemerin affect metabolic status and reproductive function in many species. The hypothesis in the present study was that there were chemerin mRNA transcript and protein in the pituitary of pigs and that relative abundances fluctuate during the estrous cycle and early pregnancy. Chemerin is thought to modulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion during the estrous cycle. Changes in the relative abundance of chemerin mRNA transcript and protein in anterior (AP) and posterior (PP) pituitaries of pigs were investigated, for the first time in the present study, during four phases of the estrous cycle and four periods of early pregnancy. Chemerin protein was localized in gonadotrophs, thyrotrophs and somatotrophs during the estrous cycle and early gestation. Chemerin treatments affected both basal, GnRH- and/or insulin-induced LH and FSH production, with there being variations with phase of the estrous cycle when tissues were collected. These findings indicate chemerin may be produced locally in the pituitary and may affect female reproductive function by controlling the release of LH and FSH from AP cells.

Loci associated with conception rate in crossbred beef heifers.

The inability of beef cattle to maintain full term pregnancies has become an economic concern for the beef industry. Herd management and nutritional improvements have alleviated environmental impacts on embryonic and fetal loss, yet additional gains can be made through genomic selection. The objectives of this study were to identify loci and gene-sets in crossbred beef heifers associated with the number of services required to become pregnant (TBRD) and heifer conception rate at first service (HCR1). Heifers (n = 709) from a commercial beef operation underwent one round of artificial insemination, before exposure to bulls for natural service for 50 days. Pregnancy and time of conception was determined by ultrasound 35 days after the breeding season. Heifers were genotyped using the GeneSeek (Lincoln, NE) Bovine GGP50K BeadChip prior to genome-wide association analyses (GWAA) conducted using an EIGENSTRAT-like model to identify loci associated (P < 1 × 10-5) with TBRD and HCR1. One locus was associated (P = 8.97 × 10-6) with TBRD on BTA19 and included the positional candidate gene ASIC2, which is differentially expressed in the endometrium of fertility classified heifers, and the positional candidate gene, SPACA3. Gene-set enrichment analyses using SNP (GSEA-SNP) data, was performed and identified one gene-set, oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen as enriched (NES = 3.15) with TBRD and contained nine leading edge genes that contributed to the enrichment of the gene set. The enriched gene-set is involved in catalyzing oxidation-reduction reactions, which have been associated with oxidative stressors impacting pregnancy success. No loci were associated nor gene-sets enriched with HCR1. Identification of loci, positional candidate genes, gene-sets and leading edge genes enriched for fertility facilitate genomic selection that allows producers to select for reproductively superior cattle, reduce costs associated with infertility, and increase percent calf crop.

Transcriptome profiling of buffalo endometrium reveals molecular signature distinct to early pregnancy.

Buffalo reproduction struggles with a high incidence of early embryonic mortality. Effective treatment and prevention strategies for this condition are not available due to lack of understanding of molecular pathways in early pregnancy of this species. In the present study, we have attempted to understand these molecular pathways by characterizing the endometrial transcriptomic profiles of pregnant buffalos during early pregnancy. For the transcriptome profiling, buffalo endometrial tissues of 29-36 days of pregnancy and of nonpregnant luteal phase were collected from the local slaughterhouse. We confirmed the status of pregnancy based on the crown vertebral length of the foetus. Total RNA was isolated and sequencing was performed using the Illumina nextseq platform. The raw reads were filtered and mapped to the Bos taurus UMD 3.1 reference genome assembly. An average of 24,597 genes was investigated for differential expression between the two groups. Transcriptome data identified a total of 450 differentially expressed genes (using a cut off value of log2 fold changes >2 and <-2) in early pregnancy in comparison to the nonpregnant group (Padj < 0.05). Among these, 270 genes were significantly upregulated and 180 genes were downregulated. The most impacted pathways were related to secretion, transport, ionic homeostasis, mitosis and negative regulation of viral processes. In conclusion, our study characterized a unique set of DEGs, during the early pregnancy of buffalo, which potentially modulate the endometrial environment to establish and maintain a successful pregnancy.

Changes in the expression and distribution of junction and polarity proteins in the porcine endometrium during early pregnancy period.

The maternal endometrium undergoes transformations during early pregnancy period to regulate the paracellular permeability across the epithelium and to enable adhesion between the trophoblast and endometrial epithelial cells. These transformations, under the influence of ovarian hormones, are associated with a partial loss in polarity of epithelial cell that is regulated by tight junctions (TJ), adherens junctions (AJ) and associated polarity protein complexes. This study examined the change in expression and distribution of proteins associated with TJs, AJs and apical partition defective (PAR) complex in porcine endometrium on Days 10, 13 and 16 of estrous cycle and pregnancy. Moreover, effect of hormones, progesterone (P4) and 17-ß estradiol (E2) on polar phenotype of endometrial epithelial cells was also investigated in vitro. There was pregnancy induced increase in gene and protein expression of TJ associated claudin-1 (CLDN1) on Day 13 of pregnancy as compared to corresponding day of estrous cycle and a decrease in TJ protein, zona occludens-1 (ZO-1) and PAR complex associated PAR6 expression levels on Day 16 of pregnancy (P < 0.05). Immunofluorescence studies revealed that on Days 10 and 13, TJ proteins occludin (OCLN) and ZO-1were primarily present in the apical region of lateral epithelial membrane. On Day 16 of pregnancy, whereas, OCLN redistributed into cytoplasm, ZO-1 decreased apically but was found to localize in the basal epithelium. The AJ proteins cadherin and ß-catenin were located at the apical epithelium on Day 10 of estrous cycle and pregnancy and Day 13 of estrous cycle. On Days 13 and 16 of pregnancy both proteins were expressed in the lateral membrane and co-localization between these proteins was observed on Day 16. On Day 10, PAR complex proteins PAR3, cell division control protein 42 (CDC42) and atypical protein kinase C (aPKC) ζ were observed in apical epithelium and in lateral membrane and CDC42 was also present in the cytoplasm of epithelium. Pregnancy induced redistribution of aPKCζ to cytoplasm and CDC42 to apical surface of luminal epithelium was observed on Days 13 and 16. The in vitro P4 and E2 treatment of epithelial cells mimicked in vivo results. These results indicate that P4 and E2 regulate alterations in epithelium that may facilitate embryo implantation and given the role of cadherin, catenin and CDC42 in embryo invasion, change in distribution of these proteins may limit the invasiveness of porcine conceptuses into the stroma.

Molecular characterization and expression profile of interferon-stimulated gene 15 (ISG15) in the endometrium of goat (Capra hircus).

Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is responsible for uterine receptivity, implantation and conceptus development in different ruminant species, but in goat (Capra hircus) its role is yet to be explicated. In the present study, the ISG15 gene was cloned, characterized and its temporal expression profile was examined in the endometrium of caprine (cp). A fragment of cpISG15 gene, 1033 bp in length, was amplified, cloned and sequenced from genomic DNA covering the coding region. Sequence analysis of cpISG15 gene revealed that it was comprised of two exons of 59 bp and 496 bp encoding a peptide of 157 amino acids. Complementary DNA (cDNA) and deduced amino acid sequences of cpISG15 exhibited 99 and 98, 93 and 88, 94 and 89, 76 and 66, and 73 and 62% identity with that of sheep, cattle, buffalo, human and mice, respectively. Further, relative expression of cpISG15 mRNA and protein was determined by quantitative real-time PCR (qPCR) and Western blot, respectively, in the endometrium of pregnant and cyclic does. Both cpISG15 mRNA and protein were expressed maximally (P < 0.05) in the endometrium during early stage of pregnancy (16-24 d) as compared to cyclic does, but no significant difference was observed in cpISG15 mRNA and protein expression in the endometrium between the later stage of pregnancy (25-40 d) and cyclic does. It is concluded that cpISG15 is almost similar in structure and probably in function also to other species as it has been found significantly upregulated during early pregnancy.

Maternal plasma proteomics in a rat model of pregnancy complications reveals immune and pro-coagulant gene pathway activation.

PROBLEM: The Brown Norway (BN) rat is a model of T-helper 2 immune diseases, and also a model of pregnancy disorders that include placental insufficiency, fetal loss, and pre-eclampsia-like symptoms. The aim of this study was to investigate the plasma proteomic/cytokine profile of pregnant BN rats in comparison to that of the Lewis (LEW) rat strain. METHOD OF STUDY: Plasma proteomics differences were studied at day 13 of pregnancy in pooled plasma samples by differential in-gel electrophoresis, and protein identification was performed by mass spectrometry. Key protein findings and predicted cytokine differences were validated by ELISA using plasma from rats at various pregnancy stages. Proteomics data were used for ingenuity pathway analysis (IPA). RESULTS: In-gel analysis revealed 74 proteins with differential expression between BN and LEW pregnant dams. ELISA studies confirmed increased maternal plasma levels of complement 4, prothrombin, and C-reactive protein in BN compared to LEW pregnancies. LEW pregnancies showed higher maternal plasma levels of transthyretin and haptoglobin than BN pregnancies. Ingenuity pathway analysis revealed that BN pregnancies are characterized by activation of pro-coagulant, reactive oxygen species, and immune-mediated chronic inflammation pathways, and suggested increased interleukin 6 and decreased transforming growth factor-ß1 as potential upstream events. Plasma cytokine analysis revealed that pregnant BN dams have a switch from anti- to pro-inflammatory cytokines with the opposite switch observed in pregnant LEW dams. CONCLUSION: Brown Norway rats show a maternal pro-inflammatory response to pregnancy that likely contributes to the reproductive outcomes observed in this rat strain.

Differential expression of Sox9 protein and proteoglycans in the epiphyseal cartilage of bromocriptine-treated pregnant and lactating rats.

Several investigations have shown that pregnancy and lactation are able to induce elongation of long bone by altering epiphyseal cartilage function in a prolactin-dependent manner. Since the transcription factor Sox9 is of utmost importance for chondrocyte proliferation and differentiation and since bromocriptine, a dopaminergic D2 agonist widely used to suppress milk production, is known to disrupt the production and release of prolactin, we herein aimed to investigate whether pregnancy and lactation as well as bromocriptine could alter the expression of Sox9. Our immunohistochemical analysis showed that the Sox9 expression levels were markedly upregulated in the tibial proliferative zone of day 21 pregnant rats. In day 8 (early) and day 14 (mid) lactating rats, the Sox9 expression was enhanced only in the proliferative zone, but not in the resting and hypertrophic zones. There was no change in Sox9 expression in day 21 (late) lactating rats. Postweaning rats manifested a decreased Sox9 expression in the hypertrophic zone. Bromocriptine had no effect on Sox9 expression in the proliferative zone of day 21 pregnant rats; however, it completely prevented the Sox9 upregulation in those of early and mid-lactating rats. A differential response was observed in the proliferative and hypertrophic zones of late lactating rats, in which bromocriptine enhanced Sox9 expression. Further investigation of cartilaginous matrix revealed no change in proteoglycans accumulation in lactating rats. In conclusion, the upregulated Sox9 expression predominantly occurred in the proliferative zone during late pregnancy and early and mid-lactation, while the bromocriptine effects depended on the periods and epiphyseal zones.

The effect of pregnancy on nitrogen retention, maternal insulin sensitivity, and mRNA abundance of genes involved in energy and amino acid metabolism in gilts.

Twenty-one of each pregnant (P) and nonserviced, nonpregnant (NP) sister-pairs of gilts were selected to investigate the effect of pregnancy on protein deposition (Pd; whole body and maternal), insulin sensitivity, and mRNA abundance of genes involved in energy and AA metabolism. Between breeding (study day 0) and day 111, P and NP gilts received 2.16 kg of the experimental diet (3.34 Mcal ME/kg, 17.6% crude protein, 0.78% standardized ileal digestible lysine) that was formulated to meet the estimated ME requirements of pregnant gilts (and meet or exceed AA requirements). Nitrogen balances were conducted on day 63 and 102 ± 0.2 of the study during 4-d periods. Blood samples were collected on day 43, 56, 71, 85, 98, and 108 ± 0.3 of the study to determine plasma concentrations of fasted IGF-1, estradiol (E2), and estrone sulfate (E1S). Frequently sampled intravenous glucose tolerance tests (FSIGTT) were conducted on day 75 ± 0.7 in 6 P and 5 NP gilts and on day 107 ± 0.4 in 17 P and 17 NP gilts and the MINMOD approach was applied to evaluate whole body insulin sensitivity and pancreatic responsiveness. Longissimus muscle (LM) and s.c. adipose tissue (AD) samples were excised from 12 P and 12 NP gilts at day 111 ± 0.4 of the study after euthanasia to determine mRNA abundance of key genes. Whole body Pd was greater (P < 0.001) at day 102 and maternal Pd was lower (P < 0.002) at day 63 and 102 for P compared to NP gilts. Plasma concentrations of E1S and E2 increased (P < 0.05) with study day for P gilts and remained constant for NP gilts, which coincided with reduced plasma concentrations of IGF-1 and increased estrogen receptor alpha (ESR1) mRNA abundance in LM of P gilts. Glucose effectiveness was not different between P and NP gilts, but whole body insulin sensitivity was lower (P = 0.004) in P compared to NP gilts on day 75 and 107, which corresponded with reduced mRNA abundances of SLC2A4, HK2, SREBF1, and FASN, and increased abundances of PDK4 and PPARGC1A in LM and AD. When fed identically, P gilts had greater whole body Pd at day 102, which reflects Pd in the pregnancy-associated tissues (at the expense of maternal Pd), likely driven by estrogen-stimulated insulin resistance in peripheral tissue and subsequent modulation of gene expression relating to glucose metabolism.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.