Replacing Soybean Meal with Sesame Meal in the Diets of Lactating Awassi Ewes Suckling Single Lambs: Nutrient Digestibility, Milk Production, and Lamb Growth.

Two experiments were conducted to assess the influence of sesame meal (SM) feeding on nutrient digestibility, N balance, milk production and composition, ewes' body weight change, and growth performance of lambs. In experiment 1, 18 ewe lambs were randomly distributed into three diets to evaluate the effects of soybean meal replacement with SM on nutrient intake, digestibility, and N balance. Treatments were no SM (SM0), 7.5% SM (SM7.5), or 15% SM (SM15) of the dietary dry matter (DM). Aside from intake and digestibility of ether extract (EE), which was greater in the SM-containing diets compared with SM0, intake and digestibility of the remaining nutrients was similar among dietary treatments. In experiment 2, 30 ewes suckling single lambs were randomly assigned to the same diets used in experiment 1. Intakes of DM, crude protein, neutral detergent fiber, and acid detergent fiber were unaffected by treatment. Milk yield was greater in SM diets than in the SM0 diet. Cost/kg of milk production decreased while feed efficiency improved in the SM-diets compared to the SM0 diet. In conclusion, results of the current studies demonstrate the possibility of replacing soybean meal with sesame meal in diets of lactating Awassi ewes.

The effects of Saccharomyces cerevisiae supplementation on intake, nutrient digestibility, and rumen fluid pH in Awassi female lambs.

AIM: The aim of this study was to evaluate the effect of feeding low (LO)- or high (HI)-fiber diets supplemented with Saccharomyces cerevisiae (SC) on nutrient intake, digestibility, nitrogen balance, rumen fluid pH, and serum concentrations of glucose and urea nitrogen in Awassi female lambs in a 2×2 factorial arrangement of treatments. MATERIALS AND METHODS: Experimental diets were as follows: (1) LO-fiber diet with no SC supplementation (-LO), (2) LO-fiber diet supplemented with SC (+LO), (3) HI-fiber diet with no SC supplementation (-HI), or (4) HI-fiber diet supplemented with SC (+HI). Eight female lambs were used in a replicated 4×4 Latin square design with 15-day experimental periods (10-day adaptation period and 5-day collection period). RESULTS: A fiber×SC interaction (p≤0.05) was detected for dry matter (DM) and crude protein (CP) intake among diets showing greater DM and CP intake for +LO diet compared to +HI group supplemented with SC, whereas -LO and -HI were intermediate. A fiber×SC interaction (p=0.05) was also detected for the neutral detergent fiber (NDF) intake among diets. Intake of NDF was greater for the -HI diet compared with +LO and -LO diets. Similarly, NDF intake was greater for +HI diet than -LO diet. A tendency (p=0.07) for a fiber×SC interaction was detected for acid detergent fiber (ADF) intake among diets as well. ADF intake tended to be greater for HI-fiber diets. No difference was observed in the rumen fluid pH for lambs fed with the different diets. No fiber×SC interactions were detected for the digestibility of DM, CP, NDF, and ADF among dietary treatments. Digestibility of DM was greater (72.9 g/100 g vs. 67.1 g/100 g; p=0.0002) for LO versus HI fiber. However, NDF and ADF digestibilities were greater (60.8 and 61.9 g/100 g vs. 55.8 and 52.7 g/100 g for NDF and ADF digestibility, respectively; p≤0.01) for the HI-fiber than the LO-fiber diets. CONCLUSION: Results obtained in the current study indicate that SC supplementation has a minimal effect on the performance of Awassi female lambs fed with varying fiber levels.

Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk.

Exosomes and microvesicles are extracellular vesicles released from cells and can contain lipids, miRNAs and proteins that affect cells at distant sites. Recently, microvesicles containing miRNA have been implicated in uterine microenvironment of pigs, a species with unique epitheliochorial (non-invasive) placentation. Here we report a novel role of conceptus-derived exosomes/microvesicles (hereafter referred to as extracellular vesicles; EVs) in embryo-endometrial cross-talk. We also demonstrate the stimulatory effects of EVs (PTr2-Exo) derived from porcine trophectoderm-cells on various biological processes including the proliferation of maternal endothelial cells (PAOEC), potentially promoting angiogenesis. Transmission immuno-electron microscopy confirmed the presence of EVs in tissue biopsies, PTr2-Exo and PAOEC-derived EVs (PAOEC-Exo). RT-PCR detected 14 select miRNAs in CD63 positive EVs in which miR-126-5P, miR-296-5P, miR-16, and miR-17-5P were the most abundant angiogenic miRNAs. Proteomic analysis revealed EV proteins that play a role in angiogenesis. In-vitro experiments, using two representative cell lines of maternal-fetal interface, demonstrated bidirectional EVs shuttling between PTr2 and PAOEC cells. Importantly, these studies support the idea that PTr2-Exo and PAOEC-Exo containing select miRNAs and proteins can be successfully delivered to recipient cells and that they may have a biological role in conceptus-endometrial cross-talk crucial for the pregnancy success.

Altered expression of chemokines and their receptors at porcine maternal-fetal interface during early and mid-gestational fetal loss.

Chemokines play a significant role in pregnancy, especially during embryonic attachment and placental development. During early pregnancy, immune cells are recruited extensively to the endometrium in several species including pigs. However, this recruitment is solely mediated by the presence of the conceptus in pigs making it a unique feature compared with other species (humans, primates and mice). To understand the biological significance of chemokine expression and immune cell recruitment in the context of fetal loss, we investigate a well-characterized porcine fetal loss model during the window of early pregnancy at gestational day (gd) 20 and mid-pregnancy (gd50). These periods coincide with 25-40 % of conceptus loss. Using targeted quantitative polymerase chain reaction and Western blot approaches, we screened a specific set of chemokines. Comparisons were made with endometrial lymphocytes (ENDO LY), endometrium and chorioallantoic membranes (CAM) associated with spontaneously arresting and healthy conceptus attachment sites (CAS). mRNA expression studies revealed an increased expression of CXCR3 and CCR5 in ENDO LY and of CXCL10, CXCR3, CCL5 and CCR5 in the endometrium associated with arresting CAS at gd20. DARC was decreased in the endometrium at gd50. CCL1 was increased in CAM associated with arresting CAS at gd50. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5 and CCR5) in the endometrium and CAM. CD45+ immunohistochemistry demonstrated a significantly higher localization in ENDO LY in the endometrium associated with healthy versus arresting counterparts. Most of these differences were observed in early pregnancy and might contribute towards a shift in immune cell functions.

Intrinsic determinants and predictors of superovulatory yields in sheep: Circulating concentrations of reproductive hormones, ovarian status, and antral follicular blood flow.

Hormonal ovarian superstimulation has contributed to small ruminant reproduction around the world, impacting genetic improvement and zoosanitary programs, contributing to the conservation of endangered species, and supporting other related biotechnologies. Advanced knowledge surrounding the superovulatory treatments in sheep has resulted in enhanced control of influencing factors and improved the protocols currently used. However, in spite of minimization of some adverse factors, superovulatory responses in ewes still remain variable, preventing the more widespread use of superovulation in commercial embryo transfer programs and reproductive research in this species. Recent evidence demonstrates that changes in antral follicular populations and blood supply, and circulating concentrations of certain reproductive hormones determined at the specific time points just before or during the superovulatory treatment are associated with superovulation success in ewes. This review attempts to compile the data from available literature to identify ovarian and hormonal determinants of the superovulatory outcome in ewes, which can be used to substantially improve the existing protocols and to reduce the extra cost and unnecessary stress imposed on poorly responding animals. An overview of most commonly used and some recently developed, FSH-based ovarian stimulation protocols is given at the outset to highlight variation in the frequency and timing of gonadotropin injections, estrus synchronization methods, and follicular wave synchronization and/or ovulation induction techniques during the superovulatory treatments in ewes.

Placentation, maternal-fetal interface, and conceptus loss in swine.

Pregnancy is a delicate yet complex physiological process that requires fine-tuning of many factors (hormones, growth factors, cytokines, and receptors) between the mother and the conceptus to ensure the survival of the conceptus(es) to term. Any disturbance in the maternal-conceptus dialog can have detrimental effects on the affected conceptus or even the outcome of pregnancy as a whole. Being a litter-bearing species, such disruptions can lead to a loss of up to 45% of the totally healthy offspring during early (periattachment) and midgestation to late gestation in pigs. Although the exact mechanism is not entirely understood, several factors have been associated with the fetal loss including but not limited to uterine capacity, placental efficiency, genetics, nutrition, and deficits in vascularization at the maternal-fetal interface. Over the years, we investigated how immune cells are recruited to the porcine maternal-fetal interface and whether they contribute to vascularization. We also delineated how cytokines, chemokines, and cytokine destabilizing factors fine-tune inflammation and whether the cytokine shift from early to midpregnancy exists at the porcine maternal-fetal interface. Finally, we evaluated the role of microRNAs in regulating immune cell recruitment and their angiogenic functions during pregnancy. Collectively our research points out that the immune-angiogenesis axis at the porcine maternal interface is significantly involved in promoting new blood vessel development, regulating inflammatory responses and ultimately contributing to pregnancy success. In this review, we summarized current knowledge on spontaneous fetal loss in swine, with special attention to the mechanisms in immune reactivity and interplay at the maternal-fetal interface.

Comparison of the effects of pretreatment with Veramix sponge (medroxyprogesterone acetate) or CIDR (natural progesterone) in combination with an injection of estradiol-17ß on ovarian activity, endocrine profiles, and embryo yields in cyclic ewes superovulated in the multiple-dose Folltropin-V (porcine FSH) regimen.

Follicular wave status at the beginning of exogenous FSH administration is an important contributor to variability in superovulatory responses in ruminants. Studies in ewes have shown a decrease in the number of ovulations when superovulation is initiated in the presence of ostensibly ovulatory-sized ovarian follicles. Hormonal ablation of large antral follicles with the progestin-estradiol (E2-17ß) treatment significantly reduces this variability in superovulated anestrous ewes, but the effects of the treatment in cycling ewes have not yet been assessed. Sixteen Rideau Arcott × Polled Dorset ewes (November-December) received either medroxyprogesterone acetate (MAP)-releasing intravaginal sponges (60 mg) or controlled internal drug release (CIDR) devices (containing 300 mg of natural progesterone) for 14 days (Days 0-14), with a single intramuscular injection of 350 µg of E2-17ß on Day 6. The superovulatory treatment consisted of six injections of porcine FSH (Folltropin-V) given twice daily, followed by a bolus GnRH injection (50 µg intramuscular) on Day 15. There were no differences (P < 0.05) in the ovulatory responses and embryo yields between the two groups of ewes. In both subsets of animals, the next follicular wave emerged ∼2.5 days after an E2-17ß injection (P > 0.05). A decline in maximum follicle size after an E2-17ß injection was more abrupt in CIDR- compared with MAP-treated animals, and the ewes pretreated with exogenous progesterone had significantly more 3-mm follicles at the start of the superovulatory treatment. The metabolic clearance rate of exogenous E2-17ß appeared to be greater in MAP-treated ewes, but circulating concentrations of porcine FSH failed to increase significantly after each Folltropin-V injection in CIDR-treated animals. The CIDR-treated ewes exceeded (P < 0.05) their MAP-treated counterparts in serum E2-17ß concentrations during superovulation. In spite of differences in antral follicle numbers and endocrine profiles between MAP- and CIDR-treated cyclic ewes receiving E2-17ß before ovarian superstimulation, there were no differences in superovulatory responses.

Distinct microRNA expression in endometrial lymphocytes, endometrium, and trophoblast during spontaneous porcine fetal loss.

Endometrial lymphocytes are recruited to the porcine maternal-fetal interface by conceptus-derived signals. The transiently recruited lymphocytes adopt a specialized phenotype in the endometrium that regulates various placental physiological processes, including angiogenesis. Small non-coding RNAs, microRNAs (miRNAs) are emerging as principal bio-molecules regulating the development of lymphocytes and their angiogenic functions. However, no information is available in the context of endometrial lymphocytes in pregnancy. We hypothesize that miRNAs are involved in the development of endometrial lymphocytes and their angiogenic functions at the porcine maternal-fetal interface. Using a targeted Q-PCR approach for selected miRNAs involved in immune cell development, angiogenesis, and anti-angiogenesis, we conducted a study to screen endometrial lymphocytes associated with healthy and spontaneously arresting conceptus attachment sites (CAS) at two well-defined periods of fetal loss. Comparisons were made with endometrium and trophoblasts associated with healthy and arresting CAS. In addition, levels of putative mRNA targets and subsequent functional clustering of genes were studied in order to predict the biological mechanisms affected. We found several significant differences for miRNAs involved in immune cell development and angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between endometrial lymphocytes associated with healthy and arresting CAS. Significant differences were also found in endometrium and trophoblasts for some miRNAs (miR-20b, miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, selected mRNA targets showed differential expression in all groups. Our data, although associative, are the first to unravel the selected miRNAs involved in immune cell development and provide insights into their possible regulation in abortive pregnancy.

mRNA destabilizing factors: tristetraprolin expression at the porcine maternal-fetal interface.

PROBLEM: To evaluate the expression of the tristetraprolin family and their selected targets during porcine pregnancy. METHOD OF STUDY: Using qPCR and Western blot, mRNA and protein levels were compared between endometrium and chorioallantoic membrane (CAM) associated with healthy and impaired conceptuses at gestation day (gd) 20 and gd50, respectively. Immunohistochemistry was performed to determine localization of TIS11 family members at gd20 and 50. RESULTS: Multiple significant differences (P < 0.05) in TIS11 family transcripts were observed in the aforementioned comparisons. GM-CSF was significantly higher in healthy endometrium and CAM from impaired conceptus attachment sites. TNF-α was elevated in CAM as compared to endometrium at gd50, regardless of conceptus health status. Immunohistochemical staining shows TIS11 family expressed in the glandular and luminal epithelium, as well as stromal cells in the uterus. CONCLUSIONS: The shift in the expression of tristetraprolin (TTP) and TIS11D points to a potential role of these genes in regulating spontaneous fetal loss.

Expression of leptin and its long form receptor at the porcine maternal-fetal interface: contrasting healthy and arresting conceptus attachment sites during early and mid-pregnancy.

BACKGROUND: It is well established that spontaneous conceptus loss in swine is associated with an imbalance of both angiogenic and immunological factors. Leptin (LEP), a metabolic hormone, has also been implicated in the promotion of angiogenesis. In this study, LEP and its long form receptor (OB-Rb) were evaluated during porcine pregnancy to assess their basal level of expression and their potential role in conceptus development. METHODS: Expression and secretion of LEP and OB-Rb were quantified in the endometrium of non-pregnant (n = 5), and in the endometrium and chorioallantoic membrane (CAM) of pregnant sows (parity 2 to 5) at gestational days (gd) 20 (n = 8) and 50 (n = 8). Data were analyzed by a 3-way ANOVA testing the effects of conceptus health, tissue type and gestational day. RESULTS: Leptin and OB-Rb transcripts were significantly higher (P < 0.05) in pregnant than in non-pregnant sows. Significantly greater LEP (P < 0.001) was detected in the endometrial tissue at gd20 compared with gd50. At the protein level, the lowest LEP expression (P < 0.01) was detected in the CAM at gd50, while OB-Rb protein was significantly lower (P < 0.01) at gd50 in the CAM than in the endometrium collected from gd20 and gd50 conceptus attachment sites. Immunofluorescence staining confirmed the expression of these proteins at both gestational days and in both tissue types. CONCLUSIONS: Changes in the expression patterns of LEP and OB-Rb between gd20 and gd50 suggest a role for the LEP/OB-R complex at the early stages of porcine pregnancy, possibly affecting the attachment process. Further mechanistic studies are warranted to understand the specific role of leptin in porcine pregnancy.

The microRNAome of pregnancy: deciphering miRNA networks at the maternal-fetal interface.

MicroRNAs (miRNAs) post-transcriptionally regulate a vast network of genes by inhibiting mRNA translation. Aberrant miRNA expression profiles have been implicated in pathologies and physiological processes including pregnancy and angiogenesis. Using our established model of implantation failure and spontaneous fetal loss in pigs (Sus scrofa), 236 miRNAs were profiled and compared between 1) non-pregnant and pregnant endometrium, 2) maternal and fetal tissues, and 3) viable and growth-arrested conceptus attachment sites by microarray and Real-Time PCR. Many significant differences in miRNA expression were observed between each of the aforementioned comparisons, and several were validated by PCR. Results indicated which miRNAs were important during pregnancy, which were elevated on the maternal or fetal side of the maternal-fetal interface, and they implicated the maternal expression of miR-10a, 27a, 29c, 323, 331-5p, 339-3p, 374b-5p, and 935 in the spontaneous loss observed in pigs. Several putative mRNA targets of the miRNAs (elevated in endometrium associated with arresting conceptuses) were assessed by quantitative Real-Time PCR and were depressed, supporting their regulation by miRNAs. Finally, targets were clustered by function to obtain ranked lists of gene networks that indicated which pathways/physiological processes might be important in non-pregnant (extracellular matrix factors) versus pregnant endometrium (nuclear transcription factor regulation), maternal (blood vessel development) versus fetal (neuronal differentiation) tissue, and healthy (extracellular matrix factors) versus arresting (GRAM domain) conceptus attachment sites. Overall, we demonstrate the presence of miRNAs on both sides of the maternal-fetal interface, implicate them in spontaneous fetal loss, and present a unique glimpse into the vast microRNAome of pregnancy.

The use of melatonin and progestagen-eCG to initiate reproductive activity in prepuberal Awassi ewe lambs.

This experiment was conducted to evaluate the effect of administering hormonal treatments (melatonin and progestagen/equine chronic gonadotropin (eCG)) on advancing puberty in Awassi ewe lambs. Fifty-one 6-month-old ewe lambs of similar body weights (around 28 kg) were randomly assigned into four treatment groups; control (CON; n = 14), melatonin (MEL; n = 13), melatonin plus progestagen-eCG (MELPP; n = 11), and progestagen-eCG (PP; n = 13). Ewe lambs in the PP and MELPP groups were fitted with intravaginal progestagen sponges containing 60 mg medroxyprogesterone acetate for 14 days; 400 IU eCG were administered to each of these ewe lambs on the day of sponge removal. Ewe lambs in the MEL and MELPP groups received subcutaneous melatonin implants (Regulin®, 18 mg melatonin) 36 days before sponge insertion. Hormonal treatment had no effect on ewe lamb body weight change. Estrous behavior was greater (p < 0.001) in the PP and MELPP groups than in the CON and MEL ewe lambs. The duration from ram introduction to onset of estrus was shorter (p < 0.05) in the CON, PP, and MELPP than in the MEL ewe lambs. The number of ewe lambs showing luteal activity was greater (p < 0.001) in the MELPP and PP than in the CON and MEL groups. Lambing rate from mating during the first cycle following ram introduction differed significantly (p < 0.01) among treatments being greater in the MELPP and PP than in the CON and MEL groups. Based on these results, it can be concluded that progestagen-eCG appears to be more effective than melatonin in inducing reproductive activity. However, the majority of ewe lambs in the current study either failed to cycle or stopped cycling following the induced estrus. Such failure in advancing puberty may be related to body weight of ewe lambs at the time of mating.