The aims of this study were i) to characterize the global changes in the composition of the uterine luminal fluid (ULF) from pregnant heifers during pregnancy recognition (day 16) using nano-LC MS/MS; ii) to describe quantitative changes in selected proteins in the ULF from days 10, 13, 16 and 19 by Isobaric tags for Relative and Absolute Quantification (iTRAQ) analysis; and iii) to determine whether these proteins are of endometrial or conceptus origin, by examining the expression profiles of the associated transcripts by RNA sequencing. On day 16, 1652 peptides were identified in the ULF by nano-LC MS/MS. Of the most abundant proteins present, iTRAQ analysis revealed that RPB4, TIMP2 and GC had the same expression pattern as IFNT, while the abundance of IDH1, CST6 and GDI2 decreased on either day 16 or 19. ALDOA, CO3, GSN, HSP90A1, SERPINA31 and VCN proteins decreased on day 13 compared with day 10 but subsequently increased on day 16 (P!0.05). Purine nucleoside phosphorylase (PNP) and HSPA8 decreased on day 13, increased on day 16 and decreased and increased on day 19 (P!0.05). The abundance of CATD, CO3, CST6, GDA, GELS, IDHC, PNPH and TIMP2 mRNAs was greater (P!0.001) in the endometrium than in the conceptus. By contrast, the abundance of ACTB, ALDOA, ALDR, CAP1, CATB, CATG, GD1B, HSP7C, HSP90A, RET4 and TERA was greater (P!0.05) in the conceptus than in the endometrium. In conclusion, significant changes in the protein content of the ULF occur during the pre-implantation period of pregnancy reflecting the morphological changes that occur in the conceptus.

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Abstract. The aim of the present study was to investigate the effect of short-term progesterone (P4) supplementation during the early metoestrous period on circulating P4 concentrations and conceptus development in cattle. The oestrous cycles of cross-bred beef heifers were synchronised using a 7-day P4-releasing intravaginal device (PRIDÒ Delta; 1.55 g P4) treatment with administration of a prostaglandin F 2a analogue (Enzaprost; CEVA Sante Animale) the day before PRIDÒ Delta removal. Only those heifers recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to one of five groups: (1) control: no treatment; (2) placebo: insertion of a blank device (no P4) from Day 3 to Day 7; (3) insertion of a PRIDÒ Delta from Day 3 to Day 7; (4) insertion of a PRIDÒ Delta from Day 3 to Day 5; or (5) insertion of a PRIDÒ Delta from Day 5 to Day 7. In vitro-produced blastocysts were transferred to each heifer in Groups 2-5 on Day 7 (n ¼ 10 blastocysts per heifer) and conceptuses were recovered when heifers were killed on Day 14. Based on the outcome of Experiment 1, in Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to one of three treatment groups: (1) placebo; (2) PRID from Day 3 to Day 5; or (3) PRID from Day 3 to Day 7. All heifers were killed on Day 16 and recovered conceptuses were incubated in synthetic oviducal fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-t (IFNT). In both experiments, daily blood samples were taken to determined serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC, USA). Insertion of a PRID resulted in an increase (P , 0.05) in serum P4 that declined following removal. In Experiment 1, P4 supplementation from Day 3 to Day 5 (17.0 AE 1.4 mm) or Day 3 to Day 7 (11.3 AE 2.3 mm) increased conceptus length compared with placebo (2.1 AE 1.8 mm). Serum P4 was significantly lower from Day 9 to Day 14 (P , 0.05) and the weight of the Day 14 corpus luteum (CL) was lower in the PRID Day 3-7 group than the placebo or control groups. In Experiment 2, supplementation from Day 3 to Day 5 (94.0 AE 18.8 mm) or Day 3 to Day 7 (143.6 AE 20.6 mm) increased conceptus length on Day 16 compared with placebo (50.3 AE 17.4 mm). Serum P4 was significantly lower in the two supplemented groups following PRID removal compared with placebo (P , 0.05) and was associated with a lower CL weight in the Day 3-7 group. Conceptus length was strongly correlated with the IFNT concentration in the uterine flush (r ¼ 0.58; P ¼ 0.011) and spent culture medium (r ¼ 0.68; P , 0.002). The findings of the present study highlight the somewhat paradoxical effects of P4 supplementation when given in the early metoestrous period in terms of its positive effect on conceptus development and its potentially negative effects on CL lifespan.

The aim was to investigate the effect of lactation on the composition of pre-ovulatory follicular fluid (FF). Forty in-calf primiparous heifers and 20 maiden heifers were enrolled. Immediately after calving, half of the cows were dried off while the remainder were milked twice daily. Serum samples were collected twice weekly from two weeks pre- to 84 days postpartum (dpp). FF was analysed by gas chromatography-mass spectrometry. Serum concentrations of non-esterified fatty acids and β-hydroxybutyrate were higher, while glucose, insulin and Insulin-like growth factor 1 (IGF1) concentrations were lower in lactating cows compared with non-lactating cows and heifers (P<0.01). Principal component analysis of FF metabolites revealed a clear separation of the lactating group from both non-lactating cows and heifers. The amino acids tyrosine, phenylalanine and valine and fatty acids heneicosanoic acid and docosahexaenoic acid were all lower in FF from lactating compared with dry cows (P<0.05). FF from lactating cows was higher in aminoadipic acid, α-aminobutyric acid, glycine and serine while histidine, leucine, lysine, methionine and ornithine were all lower than in dry cows and heifers (P<0.05). The ratio of n6:n3 was higher in lactating cows compared with both non-lactating cows and heifers, whereas total n3 polyunsaturated fatty acids, pentadecanoic, linolenic, elaidic and arachidonic acids were all lower in the FF of lactating cows than both non-lactating cows and heifers (P<0.05). In conclusion, lactation induces distinct changes in the overall metabolic status of postpartum lactating dairy cows which are associated with divergent metabolite profiles in FF.