Lactation-induced changes in metabolic status and follicular-fluid metabolomic profile in postpartum dairy cows.

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 ß-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.

Proteomic analysis of uterine fluid during the pre-implantation period of pregnancy in cattle.

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.

Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: can supplementary progesterone rescue small embryos?

Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro-produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)-releasing intravaginal device (PRID) with the administration of a prostaglandin F2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro-produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%-55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm; P < 0.05), whereas insertion of a PRID from Days 3 to 5 increased conceptus length approximately fivefold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of "rescuing" poor-quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.

Ewe breed differences in fertility after cervical AI with frozen-thawed semen and associated differences in sperm penetration and physicochemical properties of cervical mucus.

The objective of this study was to examine sperm penetration through cervical mucus and associated physicochemical properties of cervical mucus from Belclare and Suffolk ewes - two breeds with divergent pregnancy rate following cervical AI using frozen-thawed semen. In Experiment 1, sperm penetration through cervical mucus was assessed in 15 Belclare and 15 Suffolk ewes at 30, 48 and 57h post sponge removal. In Experiment 2, rheological properties of mucus from 17 Belclare and 19 Suffolk ewes at 48 and 57h post sponge removal were determined. In Experiment 3, 20 Belclare and 20 Suffolk ewes were used to assess mucus ferning and pH collected at 42, 48, 57 and 65h post sponge removal. In Experiment 1, a higher number of sperm penetrated cervical mucus from Belclare ewes at 48h, reflected by a breed by time interaction (P=0.05). In Experiment 2, mucus from Suffolk ewes tended to have higher elastic and complex moduli than that from Belclare ewes (P=0.06) regardless of time of collection. There was no effect of ewe breed on the viscous modulus. In Experiment 3, there was a significant effect of time post sponge removal on ferning (P<0.01), but there was no effect of breed. There was no effect of time or breed on mucus pH. It is concluded that breed differences in the rheological properties of cervical mucus affect the ability of sperm to swim through cervical mucus and this may explain breed differences in fertility observed after cervical AI using frozen-thawed semen.