Metabolic memory determines oviductal gene expression of underfed ewes during early gestation.

Our aim was to investigate the oviduct environment by studying oviduct gene expression after undernutrition in day-5 pregnant ewes with different initial (i) BCS, and its association with the number of embryos recovered. Thirty-six ewes were divided into 2 groups with different iBCS: iBCS ≥2.75 (n = 19; high, H) and iBCS ≤2.25 (n = 17; low, L), and were randomly assigned to two nutritional treatments for 20 days: 1.5 (control, C) or 0.5 (underfed, U) times the daily maintenance requirements. Thus, the final four groups were: high-iBCS control (HC, n = 9), high-iBCS underfed (HU, n = 10), low-iBCS control (LC, n = 9) and low-iBCS underfed (LU, n = 8). Samples of oviduct were collected and the expression of target genes was quantified using real-time PCR. While high-iBCS control ewes presented more ADIPOR1 mRNA than the high-iBCS underfed group (P < 0.05) and low-iBCS control ewes (P = 0.01), high-iBCS underfed group presented higher ADIPOR2 gene expression than low-iBCS underfed ewes (P < 0.01) evidencing a differential oviductal gene expression for these receptors. In high-iBCS ewes, control animals presented higher IGFBP2 gene expression than underfed ewes (P < 0.05), associated these results with a poor oviductal environment. High-iBCS underfed ewes presented higher IGFBP4 gene expression than high-iBCS control ewes (P < 0.05). Stepwise regression models, using various combinations of data on metabolic and reproductive hormones, and oviduct gene expression as independent variables, identified a set of variables that accounted for 75% of the variation in the number of embryos recovered. In conclusion, the oviductal gene expression depends on body reserves and nutritional treatment, and the effect is gene-specific.

Local influence of the corpus luteum on the ipsilateral oviduct and early embryo development in the ewe.

The objective of this study was to evaluate the local effect of the corpus luteum (CL) on ipsilateral oviduct-uterus functionality and early embryo development in ewes. A total of 499 embryos were transferred on Day 1 after in vitro fertilization into the ipsilateral (n = 250) and contralateral oviducts (n = 249) of 13 ewes on Day 1 after ovulation (18-20 embryos per oviduct). On Day 6, their reproductive tracts were collected and their uterine horns were flushed for embryo recovery. More recovered embryos, a higher proportion of blastocysts, and more viable embryos were collected when the embryos were transferred into the ipsilateral oviducts (P < 0.05). In addition, almost five times higher P4 concentrations and significantly lower E2 concentrations, with higher P4:E2 ratio, were found in the ipsilateral than contralateral oviductal tissue (P < 0.05). Furthermore, a higher concentration of adiponectin was found in the ipsilateral uterine tissue macerates than in the contralateral side to the CL. The ipsilateral oviductal tissue had a lower expression of PGR and IGFBP5, but the transcript expression of ADIPOR1 was higher in the ipsilateral oviductal tissue. In the uterus, the mRNA expression of ESR1, IGFBP3, IGFBP5, and LEPR was higher or tended to be higher in the ipsilateral than contralateral uterine tissue. Uterine flushing fluid collected from the ipsilateral uterine horn had lower insulin concentrations than the contralateral horn, while no differences were found in the P4 and E2 concentrations. In conclusion, on Day 6 post-ovulation, P4 was elevated in the ipsilateral oviductal tissue, embryo development was advanced, and differential gene expression of PGR, ESR1, IGFBP3, IGFBP5, LEPR, and ADIPOR1 in the oviductal or uterine tissue was found between the ipsilateral and contralateral side. This study demonstrates local regulation of the ovary on the ipsilateral oviduct/uterine horn in the ewe.

Dietary restriction in sheep: Uterine functionality in ewes with different body reserves during early gestation.

The aim of this study was to characterize insulin, leptin and IGF-1 profiles after undernutrition in pregnant ewes with different initial body condition (iBCS) and to investigate embryo quality, uterine gene expression and presence and location of proteins during early gestation (day 5). Thirty-six Rasa Aragonesa ewes were divided into 2 groups with different BCS: BCS> 2.75 (high, H, n = 19) and BCS <2.25 (low, L, n = 17) and they were randomly assigned to two nutritional treatments: 1.5 maintenance (M) (control, C) or 0.5 (M) (undernourishment, U) times the daily maintenance requirements establishing four groups: high-iBCS control (HC, n = 9), high-iBCS undernourished (HU, n = 10), low-iBCS control (LC, n = 9) and low-iBCS undernourished (LU, n = 8). High-iBCS ewes presented higher concentration of IGF-1, reflecting a better metabolic status in these animals. There was a greater proportion of high-iBCS ewes presenting more than one CL (P < 0.05), and associated greater P4 plasma concentration, number of recovered embryo and a tendency for higher embryo viability rate (P = 0.13). In uterus, undernourished ewes tended to present lower P4 (P = 0.09) and higher E2 concentration (P = 0.10). Inmunostaining of uterine progesterone and estrogen receptors (PR and ERα) was not affected by iBCS and nutritional treatment. Ewes with low-iBCS tended to have more INSR mRNA, and undernourished ewes tended to have more IGFBP2 mRNA expression (P < 0.08). An interesting finding was that the uterine response to undernutrition was dependent on iBCS: a higher expression of GHR (P < 0.05) and a tendency in IGFBP5 (P = 0.09) mRNA was found in undernourished than control ewes but only in the high-iBCS group. In summary, the present study demonstrates that the endocrine response and the uterine gene expression to undernutrition depend on the initial body energy reserves (iBCS) and appears to be associated with a differential embryo quality.

Undernutrition affects embryo quality of superovulated ewes.

To determine the effect of undernutrition on embryo production and quality in superovulated sheep, 45 ewes were allocated into two groups to be fed diets that provided 1.5 (control, C; n = 20) or 0.5 (low nutrition, L; n = 25) times daily requirements for maintenance, from oestrous synchronization with intravaginal sponges to embryo collection. Embryos were collected 7 days after the onset of oestrus (day 0). Low nutrition resulted in lower live weight and body condition at embryo collection (P < 0.05). Diet (P < 0.01) and day of sampling (P < 0.001) significantly affected plasma non-esterified fatty acid (NEFA) and insulin concentrations. Plasma leptin concentrations decreased on day 7 only in L ewes. A significant effect of dietary treatment (P < 0.05) and day (P < 0.0001) was observed on plasma insulin-like growth factor (IGF)-I concentrations. The number of recovered oocytes and embryos did not differ between the groups (L: 15.4 ± 0.4; C: 12.4 ± 0.4). Recovery rate was lower (P < 0.05) in the L (60%) than in the C group (73%). The total number of embryos and number of viable-transferable embryos (5.0 ± 0.3 and 3.4 ± 0.3 embryos, respectively) of the L group were lower (P < 0.1) when compared with controls (8.4 ± 0.4 and 6.2 ± 0.4 embryos, respectively). Undernutrition during the period of superovulation and early embryonic development reduced total and viable number of embryos. These effects might be mediated by disruption of endocrine homeostasis, oviduct environment and/or oocyte quality.

Effect of exogenous melatonin on embryo viability and uterine environment in undernourished ewes.

The effect of exogenous melatonin on embryo viability in undernourished ewes was investigated. At lambing, 24 ewes were treated (+MEL) or not (-MEL) with a melatonin implant. After 45 days, both groups were fed to provide 1.5 (Control, C) or 0.5 (Low, L) times daily maintenance requirements, so that experimental groups were: C-MEL, C+MEL, L-MEL and L+MEL. Ewes were mated (Day 0) and on Day 5 embryos were recovered and classified according to their developmental stage and morphology. Ovaries were used for in vitro fertilization and uterine horns were processed to study progesterone and oestrogen receptor (PR and ERα) expression by inmunohistochemistry. After 21 days, groups L-MEL and L+MEL had an average weight loss of 10kg (P<0.001). Number of viable embryos per CL from L+MEL (0.50±0.2) was higher than from other groups (P<0.05). Overall, the melatonin effect was particularly evident in undernourished ewes, increasing both viability (L+MEL: 65%; L-MEL: 25%; P<0.05) and pregnancy rates (L+MEL: 66.6%; L-MEL: 16.6%; P<0.05). Neither nutrition and melatonin nor their interaction had a significant effect on the in vitro oocyte development. Melatonin treatment tended to increase the percentage of positive cells to PR in deep glandular epithelium, independently of diet (P=0.09), and the greatest staining intensity of PR was observed in the luminal and superficial glandular epithelia (P<0.0001). In conclusion, melatonin implants at lambing during the breeding season improve the viability of embryos recovered from undernourished ewes, although this effect seems not to be mediated at the oocyte competence level.

Effect of undernutrition on the uterine environment during maternal recognition of pregnancy in sheep.

The effects of pregnancy and undernutrition on endometrial gene expression were investigated in ewes fed all or half their maintenance requirements and killed on Day 14 of pregnancy or of the oestrous cycle. The endometrial expression of progesterone, oestrogen, oxytocin and interferon receptors (PR, ERalpha, OXTR and IFNAR, respectively), cyclo-oxygenase (COX)-2, proliferating cell nuclear antigen (PCNA), insulin-like growth factors (IGF)-I and -II, and IGF-1 receptor (IGF-1R) was studied by immunohistochemistry or real-time reverse transcription-polymerase chain reaction. The luminal epithelium of cyclic control ewes was devoid of PR staining and had relatively high levels of ERalpha, OXTR, COX-2 and IFNAR2. The presence of a conceptus decreased the in vitro uterine secretion of prostaglandin (PG) F(2alpha) and the expression of IFNAR2 in most cell types, and increased the gene expression of IGF-I and IGF-II. Undernutrition tended to increase ERalpha protein and gene, but decreased in vitro uterine secretion of PGE(2) and the gene expression of IFNAR2 in cyclic ewes. There was no effect of undernutrition on pregnancy rates or the number of conceptuses recovered. Consistent with this, undernutrition of pregnant ewes did not have any effect on uterine gene expression. Moreover, in cases where changes were observed in cyclic ewes, these changes were negated when a conceptus was present.