F. Paradis

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = -0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = -0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.

Pig preovulatory oocytes modulate cumulus cell protein and gene expression in vitro.

This study investigated the changes in protein and gene expression in oocytectomized cumulus cells (OOX) of medium-sized follicles from gilts, cultured with or without denuded oocytes isolated from large oestrogenic sow follicles. Proteomic analysis identified 14 proteins that were differentially expressed in OOX, of which the protein 14-3-3 eta, a signal transduction pathway modulator, was down-regulated in the presence of oocytes. Oocyte co-culture also down-regulated FSHR mRNA expression in OOX, as measured by real-time PCR, and FSHR and 14-3-3 eta mRNA abundance were positively correlated. The oocyte also up-regulated HSD3B mRNA, suggesting an effect on cumulus cell progesterone synthesis. Together with data on gene expression in granulosa cells during the follicular phase of the sow oestrous cycle, this study suggests that modulation of the expression of steroidogenesis related proteins and genes in cumulus cells by the porcine preovulatory oocyte reflects the specific physiological requirements of the preovulatory follicle.

Restricted feed intake in lactating primiparous sows. II. Effects on subsequent litter sex ratio and embryonic gene expression

Expression of panels of candidate genes controlling myogenesis, angiogenesis and gender-specific imprinting of development were analysed in embryonic, placental and endometrial tissues recovered at Day 30 of gestation from a subset of primiparous sows that were either feed restricted (Restrict; n=17) or fed to appetite (Control; n=15) during the last week of the previous lactation. Embryos were also sex typed to investigate gender bias in response to treatments. Average embryonic weight was lower in the subset of Restrict compared with Control litters (1.38±0.07vs 1.59±0.08g, respectively) and the male:female sex ratio was higher (P<0.05) in embryos (litters) recovered from Restrict sows. Treatment affected (P≤0.05) the expression of embryonic and placental genes involved in insulin-like growth factor (IGF) 2 signalling, including IGF2, INSR and IGF2R. Embryonic expression of ESR1 was also affected by treatment (P<0.03) and sex×treatment interactions were observed for the expression of embryonic ESR1 (P<0.05) and placental ANGPT2 (P<0.03). At the molecular level, these results support the suggestion that changes in placental function are not the primary mechanism mediating detrimental effects of previous sow catabolism on early embryonic development in the feed-restricted lactational sow model. However, perturbations in the IGF2 system are implicated as mediators of these effects.

Ontogeny of metabolic effects on embryonic development in lactating and weaned primiparous sows

Using an established experimental paradigm, feed restriction during the last week of lactation in primiparous sows reduces embryonic growth and development and produces female-specific embryonic mortality by Day 30 of gestation. Because this gender-specific loss of embryos at Day 30 was associated with changes in the variation of markers of epigenetic imprinting, the present study sought to establish the ontogeny of such epigenetic affects. Leucocyte DNA of restrict-fed sows exhibited decreased global methylation during the last week of lactation and during the return to oestrus (P < 0.05), but no associated changes in plasma folate and vitamin B(12). Furthermore, no changes in methylation of blastocyst DNA, embryonic sex ratios or development were evident at Day 6 of gestation that would characterise the underlying defects that reduced female embryo survival by Day 30. However, regardless of treatment, embryo recovery rates and synchrony in embryonic development were associated with the stage of development of the recovered embryos (r = 0.68; P < 0.001). The subset of sows classified as bearing litters with superior embryonic development had lower net energy balance over lactation (P < 0.01) and higher ovulation rates (P < 0.005) compared with sows classified as having poorer embryonic development. Collectively, these data suggest that a subset of litters within restrict-fed sows will be most sensitive to the latent epigenetic mechanisms that ultimately trigger gender-specific loss of embryos by Day 30 of gestation, but that these selective mechanisms are not evident by Day 6 of gestation.

Temporal candidate gene expression in the sow placenta and embryo during early gestation and effect of maternal Progenos supplementation on embryonic and placental development

The present study characterised gene expression associated with embryonic muscle development and placental vascularisation during early gestation in the pig and examined effects of Progenos supplementation in early pregnancy. Tissues were collected from commercial multiparous sows (n = 48) from Days 16 to 49 of gestation. In the placenta, qPCR revealed that vascular endothelial growth factor (VEGFA) expression did not change from Day 17 to 49 of gestation; however, KDR receptor and angiopoietin-1 and -2 expression were differentially regulated, with periods of high expression corresponding to two critical phases of angiogenesis in the pig. In the embryo, the pattern of myogenesis-related gene expression was consistent with available literature. A commercially available nutritional supplement Progenos (20 g day⁻¹ L-arginine) added to the diet of sows from either Day 15 to 29 (P15-29; n = 33), Day 30 to 44 (n = 29) or from Day 15 to 44 (n = 76) of gestation tended to increase (P = 0.058) embryonic growth rate compared with non-supplemented controls (n = 79) and angiogenin expression was higher (P = 0.028) at Day 30 of gestation in placentae from sows on the P15-29 Progenos treatment. These results are consistent with proposed beneficial effects of l-arginine on early embryonic development and placental vascularisation.

Proteomics of dark cutting longissimus thoracis muscle from heifer and steer carcasses

Studies have suggested that the phenomenon of dark cutting (Canada B4) beef may also be related to muscle glycolytic proteins. The objective of this study, therefore, was to analyze longissimus thoracis (LT; n=23), from Canada AA (n=8), atypical (AB4; pH<5.9, n=8) and typical (TB4; pH>5.9, n=7) B4 heifer and steer carcasses, for sarcoplasmic and myofibrillar proteins using 2-D gel electrophoresis and LC-MS/MS mass spectrometry. Results indicated that AB4 LT had intramuscular pH and lactate concentration similar to Canada AA but lower (P<0.05) L* and b*. Moreover, AB4 LT were tougher than Canada AA even at 21days post-mortem, unlike TB4. Canada AB4 LT had reduced (P<0.05) levels of creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and glycerol-3-phosphate dehydrogenase [NAD(+)], indicating a compromised glycolytic capacity in AB4. Canada AB4 LT had increased (P<0.05) abundances of phosphatidylethanolamine-binding protein 1 and small heat shock proteins.

Maternal nutrient restriction in mid-to-late gestation influences fetal mRNA expression in muscle tissues in beef cattle

BackgroundManipulating maternal nutrition during specific periods of gestation can result in re-programming of fetal and post-natal development. In this experiment we investigated how a feed restriction of 85% compared with 140% of total metabolizable energy requirements, fed to cows during mid-to-late gestation, influences phenotypic development of fetuses and mRNA expression of growth (Insulin-Like Growth Factor family and Insulin Receptor (INSR)), myogenic (Myogenic Differentiation 1 (MYOD1), Myogenin (MYOG), Myocyte Enhancer Factor 2A (MEF2A), Serum Response Factor (SRF)) and adipogenic (Peroxisome Proliferator Activated Receptor Gamma (PPARG)) genes in fetal longissimus dorsi (LD) and semitendinosus (ST) muscle. DNA methylation of imprinted genes, Insulin Like Growth Factor 2 (IGF2) and Insulin Like Growth Factor 2 Receptor (IGF2R), and micro RNA (miRNA) expression, were also examined as potential consequences of poor maternal nutrition, but also potential regulators of altered gene expression patterns.ResultsWhile the nutrient restriction impacted dam body weight, no differences were observed in phenotypic fetal measurements (weight, crown-rump length, or thorax circumference). Interestingly, LD and ST muscles responded differently to the differential pre-natal nutrient levels. While LD muscle of restricted fetal calves had greater mRNA abundances for Insulin Like Growth Factor 1 and its receptor (IGF1 and IGF1R), IGF2R, INSR, MYOD1, MYOG, and PPARG, no significant differences were observed for gene expression in ST muscle. Similarly, feed restriction had a greater impact on the methylation level of IGF2 Differentially Methylated Region 2 (DMR2) in LD muscle as compared to ST muscle between treatment groups. A negative correlation existed between IGF2 mRNA expression and IGF2 DMR2 methylation level in both LD and ST muscles. Differential expression of miRNAs 1 and 133a were also detected in LD muscle.ConclusionsOur data suggests that a nutrient restriction of 85% as compared to 140% of total metabolizable energy requirements during the 2nd half of gestation can alter the expression of growth, myogenic and adipogenic genes in fetal muscle without apparent differences in fetal phenotype. It also appears that the impact of feed restriction varies between muscles suggesting a priority for nutrient partitioning depending on muscle function and/or fiber composition. Differences in the methylation level in IGF2, a well-known imprinted gene, as well as differences in miRNA expression, may be functional mechanisms that precede the differences in gene expression observed, and could lead to trans-generational epigenetic programming.