Proteomic profile in plasma as a biomarker of pregnancy in cows.

Pregnancy has its own protein dynamics, reflecting the hormonal profile. Quantitative and qualitative changes in plasma protein profile may provide useful information about this condition. Any alterations may be a signal heralding clinical or subclinical pathology. The objective of our study was to compare the plasma protein profile between selected months of pregnancy in cows for a better understanding gestation course. For this purpose, we collected blood from healthy pregnant (n = 30; n = 6 for each pregnancy stage) and non-pregnant (C; n = 6) Holstein-Friesian cows during a routine veterinary examination. Collected samples were selected according to pregnancy month (first, second, third, sixth, and ninth), prepared, and separated by two-dimensional electrophoresis. The Delta-2D program compared and statistically evaluated scanned gel images from the appropriate months. The mean volume of the spots was considered. The MALDI TOF/TOF spectrometer was used to identify statistically significant proteins. There were 11 distinct proteins found, including peptidyl-prolyl cis-trans isomerase F, oligoribonuclease, and PRELI domain-containing protein 3B (all of them have the lowest abundance in the C group), alpha-1B-glycoprotein, L-gulonolactone oxidase, hemopexin (first month with higher abundance than control), alpha-2-HS-glycoprotein (significantly higher abundance in the first month than in remaining groups), ermin (absent in the first month and lower abundance in the third and sixth months than in the remaining groups and control), endophilin-A2 (significant differences between the control and the second, third, sixth, and ninth months), apolipoprotein A-I (significant difference between control and the first and sixth months), alpha-1-antiproteinase (significant difference between control and the ninth month). The study demonstrated the distinctions between plasma protein composition and alterations during the pregnancy course which may potentially serve as diagnostic tools.

Identification of differentially expressed miRNAs in serum extracellular vesicles (EVs) of Kazakh sheep at early pregnancy.

MiRNAs-containing extracellular vesicles (EVs) possess the unique function of mediating intercellular communication and participating in many biological processes such as post-transcriptional gene regulation of embryo implantation and placental development. In the present study, Illumina small-RNA sequencing was used to identify differentially expressed (DE) miRNAs in serum EVs of pregnant (P) and non-pregnant (NP) Kazakh sheep at Day 17 from mating. The specifically and differentially expressed miRNAs at early pregnancy in sheep were verified by using RT-PCR. The target genes of DE miRNAs were predicted by bioinformatics software, and the functional and pathway enrichment analysis was performed on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. A total of 562 miRNAs (210 novel miRNAs) were identified by sequencing, of which 57 miRNAs were differentially expressed, 49 were up-regulated, 8 were down-regulated and 22 novel miRNAs were specifically expressed in the pregnant sheep. Eight highly expressed known miRNA (miR-378-3p, miR-320-3p, miR-22-3p, let-7b, miR-423-3p, miR-221, miR-296-3p, miR-147-3p) in pregnant group were down-regulated in the control group. miRNAs-containing pregnancy-related terms and regulatory pathways regulation were enriched using both GO and KEGG analyses. Moreover, we also envisioned a miRNA-mRNA interaction network to understand the function of miRNAs involved in the early pregnancy serum regulatory network. The results of RT-PCR verification confirmed the reliability of small-RNA sequencing. Among them, miR-22-3p and miR-378-3p were significantly differentially expressed (DE) between pregnant sheep and non-pregnant group (p <  0.01). The site at which oar-miR-22-3p binds MAPK3 was determined with a dual-luciferase system. This is the first integrated analysis of the expression profiles of EV-miRNAs and their targets during early pregnancy in ewes. These data identify key miRNAs that influence the implantation of sheep in the early stage of pregnancy, and provide theoretical basis for further molecular regulatory mechanisms research.

Changes in ghrelin, microminerals, antioxidants and vitamins A, E and C levels during different physiological status in high yielding Saanen goats subjected to heat stress.

The aim of this study was to evaluate the changes of ghrelin, microminerals, antioxidants, and vitamins A, E and C levels during different metabolic periods in high yielding Saanen goats subjected to heat stress. Twenty clinically and paraclinically healthy, high yielding and multiparous goats with an average age of 3 ± 0.5 years and pregnant with a single fetus were included in this study. Sampling was performed at three different physiologic periods: non-pregnancy non-lactation (P1), four-month gestation (P2), and first month of lactation (P3). In this study, the ambient temperature ranged from 19 to 42 °C and relative humidity ranged from 14 to 19% during the hot months. Serum concentrations of ghrelin, glucose (Glu), superoxide dismutase (SOD), glutathione peroxidase (GPx), vitamins (A, E and C) and microminerals (selenium, manganese, cobalt, iron, copper and zinc) were measured. Mean raw milk yield of the goats per day at the first month of lactation was 2.34 ± 0.2 kg. Concentration of ghrelin at P1 was significantly lower than P2 and P3 (P < 0.05). Glucose levels were significantly lower at P3 compared with P1 and P2 (P < 0.05). There were no significant correlations between ghrelin and Glu at different periods. Concentrations of selenium and manganese were significantly higher at P3 compared with P2 and were significantly higher at P2 compared with P1. Values of copper at P2 were significantly higher than P1 and P3 (P < 0.05). Zinc levels were significantly higher at P1 compared with P2 and P3 (P < 0.05). Values of antioxidants and vitamins were significantly lower at P3 compared with P2. It is concluded that high yielding Saanen goats may suffer from hormonal and metabolic disturbances, oxidative stress and micromineral deficiencies during late gestation and the first month of lactation especially when they are subjected to heat stress.

Serum concentrations of leptin in pregnant and non-pregnant bitches.

Leptin regulates body weight and several physiological processes including reproduction. We evaluated the circulating levels of leptin in pregnant and non-pregnant bitches as well as their correlation with body weight, food intake and number of foetuses. Nineteen healthy German shepherd bitches were used and divided in two groups (pregnant n = 12 and non-pregnant n = 7). Blood samples were collected every 15 days starting from ovulation (Day 0) throughout pregnancy (pregnant group, P) or throughout luteal phase (non-pregnant group, NP) In pregnant bitches, leptin concentrations increased from the day of ovulation (1.32 ± 0.06 ng/ml) up to day 45 (1.51 ± 0.06 ng/ml; p < .01) and returned to baseline values from day 60 post-ovulation. In non-pregnant bitches, leptin concentrations remained constant throughout the whole observation period (estimated marginal mean ± SE=1.33 ± 0.38 ng/ml). Pairwise comparisons showed significant differences between P and NP at day 45 post-ovulation (p < .05). Multivariable models indicated that, controlling for time and litter size, there was a positive relationship between leptin concentration and BW (p < .05) although Pearson coefficients showed that the correlation between BW and leptin was only significant in NP animals at day 45 (r = 0.76, p < .05). The multivariable approach also suggested that, holding BW and time constant, leptin concentrations tend to increase as the number of puppies increased (p = .06). Our study supports indirectly the contribution of the feto-placental unit to the circulating maternal leptin concentration.

Variations in serum metabolites in response to season, cyclicity, and pregnancy in estrus-synchronized Damascus goats.

The current study was carried out to determine the changes in serum electrolytes, liver enzymes, and hormones during breeding and non-breeding seasons in estrus-synchronized goats. Forty goats (n = 40) were synchronized by inserting the sponges intravaginally for 12 days; PMSG and PGF2α were injected on day of sponge removal. Blood samples were collected from the goats at the time of sponges insertion (day -12), estrus day (day 0), and at 15 day after sponge withdrawal (day 15). The blood samples were analyzed for glucose, cholesterol (CHO), aspartate aminotransferase (AST), alanine aminotransferase (ALT), thyroid stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4) and progesterone (P4), calcium (Ca), phosphate (PO4), magnesium (Mg), sodium (Na), potassium (K), and chlorine (Cl) levels. Repeated measurements ANOVA of general linear model were used as statistical model. The results showed that seasons have an effect (p < 0.05) on glucose, Ca, Na, K, Cl, CHO, T3, and T4 levels. There was an effect (p < 0.05) of cyclicity only on P4 level. Whereas, nonpregnant, or goats carrying singlet and twin fetuses have shown variation (p < 0.05) in glucose, Ca, and P4 levels. Although, the correlations existed between different serum metabolites but strong correlations (p < 0.05) were observed either between Ca and Na or T3 and T4. In conclusion, the serum electrolytes and liver enzymes are correlated with hormones during the different seasons, pregnancy status, and fecundity. In recommendation, the results of the study could be as managemental tool to monitor the reproductive activity across seasons and to maintain pregnancy carrying twining in goat breeds.

Serum and tissue pregnanes and pregnenes after dexamethasone treatment of cows in late gestation.

Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3ß-hydroxysteroid dehydrogenase (3ßHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3ßHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3ßHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3ßHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.

Comparative serum proteome analysis reveals potential early pregnancy-specific protein biomarkers in pigs.

In this study, the comparative serum proteome profile of Day 5, 12 and 16 of gestation, representing three early embryonic events, namely formation, elongation and implantation of blastocysts, and non-pregnant control were explored by a label-free quantitation-based mass spectrometric approach to identify early pregnancy biomarkers in pigs. A total of 131 proteins were identified with respect to different groups, out of which 105 were found to be differentially expressed proteins (DEPs). Among the DEPs, 54 and 66 proteins were found to be up and downregulated respectively in early pregnancy groups (fold change >2) and the maximum number of upregulated proteins was observed in the Day 12 pregnancy stage. Functional classification and pathway analysis of the DEPs revealed involvement of most of the proteins in complement and coagulation cascades, metabolic processes and immune and inflammatory responses. Proteins such as glutathione peroxidise (GPX), pregnancy zone protein (PZP), thrombospondin-1 (THBS1), α-1-antitrypsin (AAT) and mannose-binding lectin C (MBLC) were differentially expressed during early pregnancy and actively involved in different pregnancy-related activities. To the best of our knowledge, this is the first report on comparative serum protein profiling of different early pregnancy stages in pigs and our results provide a set of proteins that can be used as potential biomarkers for early pregnancy diagnosis in pigs.

Effect of prepartum exercise on lying behavior, labor length, and cortisol concentrations.

The objective of this study was to assess the effect of exercise and pasture turnout on lying behavior, labor length, and cortisol concentrations around the time of parturition in dairy cows. Twenty-nine primiparous and 31 multiparous, pregnant, nonlactating Holstein (n = 58) and Jersey × Holstein cross (n = 2) dairy cows were assigned to control (n = 20), exercise (n = 20), or pasture (n = 20) treatments at dry-off using rolling enrollment. Control cows remained in the dry cow group pen. Exercise cows were removed from the dry cow group pen 5 times per week and walked for 1.4 ± 0.1 h at 1.88 ± 0.58 km/h. Pasture cows were moved to an outdoor paddock 5 times per week for 1.8 ± 0.3 h/d. Cows were housed in deep-bedded sand freestalls in a naturally ventilated, 4-row freestall barn. Cows were moved into maternity pens on the day of projected calving or when cows displayed signs that calving was imminent (restlessness, raised or lifted tail, ruptured amniotic sac, or swollen vulva), and treatments were discontinued. Cameras continuously recorded cows from entry into the pen until farm staff noted a calf, and one observer continuously watched video for two visually observable periods throughout the calving process: time from initial observation of amniotic sac to initial observation of calf's feet, and time from initial observation of calf's feet to full expulsion of calf. Assisted calvings were excluded. Accelerometers were attached to the rear fetlocks of cows 3 d before dry-off and removed 14 d postpartum. Activity was summarized by day for the 7 d before and after delivery time recorded from video observation into lying time (hours per day), lying bout frequency (bouts per day), lying bout duration (minutes per bout), and steps (number per day). Plasma total cortisol concentration was measured on d 0 and 3 postpartum and determined by a radioimmunoassay procedure using a commercially available kit. Data were analyzed using mixed linear model. During calving, time from appearance of the amniotic sac to appearance of the calf's feet was longer for pasture cows compared with control. Control cows engaged in fewer lying bouts and less overall lying time compared with pasture and exercise cows. Cortisol concentrations were higher on the day of calving compared with 3 d later, regardless of treatment. Understanding the effects of lying alterations around calving and increases in labor period length may allow for physical activity recommendations for late-gestation dairy cows.

Relationships between body condition, body condition loss, and serum metabolites during the transition period in primiparous and multiparous cows.

In the transition period from late gestation to early lactation, dairy cows undergo tremendous metabolic changes. Insulin is a relevant antilipolytic factor. Decreasing serum concentrations of insulin and glucose, increasing serum concentrations of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB), and changes in body condition score (BCS) reflect the negative energy balance around calving. This study investigated peripartum metabolic adaptation in 359 primiparous and 235 multiparous German Holstein cows from a commercial dairy herd under field conditions. Body condition score was recorded and blood samples were taken 10 to 1 d prepartum, 2 to 4 d postpartum, and 12 to 20 d postpartum. Generalized mixed models and generalized estimation equations were applied to assess associations between prepartum BCS; BCS changes during the transition period; insulin, glucose, NEFA, and BHB serum concentrations; and milk yield, which was taken from an electronic milk meter from d 6 of lactation. Serum insulin concentrations of multiparous postpartum cows were lower compared with prepartum, and compared with primiparous cows. In general, primiparous cows had lower postpartum NEFA and BHB concentrations than multiparous cows. In primiparous cows, we identified a positive association between prepartum BCS and prepartum serum insulin concentration. Prepartum obese multiparous cows, but not primiparous cows, were characterized by higher postpartum serum NEFA and BHB concentrations and lower milk yield than other cows in the same parity class. Primiparous cows with a smaller degree of BCS loss during the transition period had higher postpartum insulin and lower NEFA concentrations and lower milk yield than other primiparous cows. In conclusion, primiparous cows had less lipolysis and lower milk yield than multiparous cows, associated with higher insulin concentrations. Avoiding high body condition loss during the transition period is a main factor in preventing peripartal metabolic imbalances of glucose and fat metabolism.

Evaluation of a bovine visual pregnancy test for the detection of pregnancy-associated glycoproteins in sheep.

As sheep produce similar pregnancy-associated glycoproteins (PAGs) to cattle, a commercially available bovine visual pregnancy test based on the detection of PAGs (visual-PAG-test) was evaluated in sheep. The test was performed with whole blood (WhB), plasma (P) and serum (S) of 163 pregnant and 153 non-pregnant ewes. Additionally, 11 pregnant ewes were tested weekly from day 14 to 49 of gestation and monthly from day 60 to day 149. Ten ewes were sampled weekly from the date of lambing until day 63 post-partum (p.p.). The sensitivity in mid-pregnancy (n = 163) was 98.16% (WhB), 99.39% (P) and 99.39% (S) compared to transabdominal ultrasonography as the gold standard while the specificity (n = 153) was 94.12% (WhB), 76.47% (P) and 93.46% (S), respectively. During early pregnancy, all 11 ewes were correctly identified as pregnant on day 42 (100%); however, the test sensitivity decreased to 54.6% (WhB) and 63.6% (S and P, respectively) at day 49. The ewes were again consistently identified as pregnant on day 63 (P) or on day 119 (S, WhB). The test was consistently negative from day 42 p.p. onwards in eight out of ten ewes. Two ewes remained consistently positive until the last sample on day 63 p.p. In conclusion, the test could be used to accurately select pregnant ewes at day 42 with a drop in sensitivity at day 49. The sensitivity of the visual-PAG-test was good in mid to late pregnancy, and early detection of pregnancy was possible in individual animals. In some ewes, the PAGs were however detectable for more than 63 days p.p.-the previous breeding history should therefore always be taken into account for correct interpretation of the test results.

Longitudinal profiles of relaxin and progestagens during pregnancy, pregnancy loss and false pregnancy in the killer whale (Orcinus orca).

The circulating pattern of immunoreactive relaxin and progestagens based on monthly and gestational stage (early, mid, late) profiles were determined during pregnancies that resulted in live calves (LIVE, n = 30), stillbirths (STILLB, n = 3), abortions (ABORT, n = 5) and presumptive false pregnancies (FALSE, n = 8), and during the follicular (n = 34) and luteal phase (n = 58). Monthly LIVE relaxin concentrations steadily increased during gestation, but values did not significantly exceed those of the luteal phase until 9 months prior to parturition, peaking during the final month at 2356 ng/ml. Relaxin surged (P < 0.05) during the final week of gestation (36,397 ng/ml), undergoing a 3 and 9-fold increase compared with concentrations in the preceding two weeks, respectively. Monthly relaxin production did not differ among each reproductive state with the exception of months-13-16 where concentrations were higher (P < 0.001) for STILLB than LIVE. Relaxin concentration was reduced (P < 0.0001) by 849% in placental versus maternal serum collected within 1 day of labor. Mid- and late-pregnancy progestagen concentrations were lower for FALSE (P < 0.001) compared with STILLB and LIVE. Late pregnancy progestagen concentrations were reduced for FALSE (P < 0.05) and ABORT (P < 0.02) compared with LIVE and STILLB. Monthly progestagen production in ABORT tended to be lower than LIVE across a range of gestational months (Months 2, 7, 8, 11) but this difference only became significant during months 14 and 15. Results indicate that relaxin is primarily produced by the CL during pregnancy, and that concentrations could not be used to differentiate from non-pregnant females until the final 6 months of gestation. In addition, as would be expected from a primarily CL product, relaxin cannot be used to detect abnormal pregnancies. Conversely, progestagens, which are produced by both the placenta and CL can be used to differentiate FALSE from normal pregnancy and may be useful indicators of fetal health in the killer whale.

Plasma calcium concentrations are decreased at least 9 hours before parturition in multiparous Holstein-Friesian cattle in a herd fed an acidogenic diet during late gestation.

Calcium homeostatic mechanisms are challenged in periparturient multiparous dairy cattle due to the rapid transport of large amounts of calcium into the mammary gland associated with colostrogenesis, resulting in decreased plasma total calcium concentration ([Ca]). An unresolved issue is the timing of the decrease in plasma [Ca] relative to the time of parturition, with the consensus view being that plasma [Ca] does not decrease until after parturition. The objective of this study, therefore, was to characterize the change in plasma [Ca] over time in periparturient dairy cattle. Plasma and mid-stream urine samples were collected daily starting 3 d before calving from 104 periparturient Holstein-Friesian dairy cows in a herd fed an acidogenic total mixed ration during the late dry period. Mixed-models ANOVA and linear and multivariable regression analyses were conducted. Plasma [Ca] decreased in periparturient multiparous cattle (n = 70) but not in primiparous cattle (n = 34). Compared with mean values approximately 72 h before parturition ([Ca] = 2.32 mmol/L), mean plasma [Ca] in multiparous cattle first decreased at 9 h before parturition (2.13 mmol/L) and remained decreased for up to 48 h after parturition, with the lowest mean value (1.87 mmol/L) occurring at 28 h after parturition. Mean 24-h urine Ca excretion was calculated to decrease by 3.5 to 3.8 g in periparturient multiparous cattle. Regression analysis indicated that plasma [Ca] in the 12-h period before and 24-h period after parturition was strongly and negatively associated with age but was also negatively associated with milk production indices. We conclude that plasma [Ca] was decreased at least 9 h before parturition in multiparous dairy cattle fed an acidogenic diet in late gestation, and that calcium homeostasis was disrupted for 2 to 3 d around parturition.

Changes in plasma concentrations of S100A7 and S100A8 in dairy cows during pregnancy.

This study was carried out to examine the changes in plasma concentrations of the Ca-binding antimicrobial proteins S100A7 and S100A8 during pregnancy in dairy cows. Holstein Friesian cows (n = 19) were inseminated with Holstein Friesian semen. Blood was collected at days 30, 60, 90, 120, 150, 180, 210, 240 and 270 after insemination. Plasma was used for measuring the concentrations of S100A7 and S100A8. Both S100A7 and S100A8 concentrations showed similar patterns during gestation; they increased during the midgestation, between days 90 and 180, and then declined before calving. The findings indicated that plasma concentrations of S100A7 and S100A8 did not change significantly during pregnancy in cows. Further studies are required to determine the roles of S100A7 and S100A8 in physiological function during pregnancy in dairy cows.

Detection of pregnancy in sheep using an ELISA for pregnancy-specific protein B.

The early detection of pregnancy and the determination of fetal numbers have economic benefits in sheep production because of the seasonal breeding patterns where missing a breeding opportunity means the loss of one productive year. The purpose of this study was to evaluate the efficacy of the B6-HRP ELISA for ovine pregnancy-specific protein B (oPSPB) measurement in the detection of pregnancy and estimation of fetal numbers in different sheep breeds. BioPRYN® ELISA assay kit was used for the detection of pregnancy in the experimental animals. Ninety-three ewes of three breeds (British Milksheep - BM, Lacaune - L and Transylvanian Racka - TR), each from three farms in Hungary, were included in the study. BM and L ewes were artificially inseminated (AI). Thirty-five days after AI, all ewes were examined by transabdominal ultrasound. The TR flock was mated naturally over a six-week period. At the end of the mating period, the ewes were similarly examined by ultrasound. Blood samples were taken from all pregnant ewes twice (35 and 65 days after AI), and serum samples were assayed by the BioPRYN test. It can be concluded that the detection of serum PSPB by ELISA is a much easier, safer, less expensive and highly accurate method for the detection of ovine pregnancy. Although some breed-related differences were detectable at 35 and 65 days post breeding, no differences in oPSPB levels were found in pregnant ewes carrying different numbers of fetuses.

Dietary supplementation for Santa Inês hair ewes on pasture at pre- and postpartum periods: dry matter intake, digestibility, milk production, and mineral metabolism.

The aim of this study was to evaluate the impact of different levels of dietary supplementation and reproductive stages on dry matter intake, digestibility, milk production, and mineral metabolism in Santa Inês hair ewes. Two dietary supplement levels of 0.5 and 1.5%, based on body weight, were used. A total of 12 hair ewes (six subjected to 0.5 and six subjected to 1.5% of concentrate supplementation based on body weight-BW) of the Santa Inês breed were evaluated in a completely randomized design with fixed effects of supplementation level, period, and its interactions. Dry matter intake, digestibility, milk production, and mineral metabolism (calcium (Ca), phosphorus (P), magnesium (Mg), alkaline phosphatase (ALP), type I insulin-like growth factor (IGF-I), parathyroid hormone (PTH), and osteocalcin (OC)) were assessed. Dry matter digestibility was affected by the supplementation level (during both pregnancy and lactation), with higher values in ewes fed at a level of 1.5% of BW. A significant interaction between treatment × reproductive stages was found for the Mg concentration. A period effect (P < 0.05) on serum concentrations of P, Ca/P, Mg, and IGF-I was observed. Serum P concentrations were influenced (P < 0.05) by treatments and reproductive stages. There were significant differences in the Ca/P ratio among the reproductive stages. The enzymatic activity of ALP and serum IGFI differed among reproductive stages. Ewes supplemented at a level of 1.5% of BW produced 18.5% more milk than ewes supplemented at a level of 0.5% of BW. The use of 0.5% of body weight in concentrate supplementation is recommended for the reduction of production costs, without having an effect on the mineral metabolism of Santa Inês hair ewes.

Circulating microRNA as candidates for early embryonic viability in cattle.

Blood-borne extracellular vesicles (i.e., exosomes and microvesicles) carrying microRNAs (miRNAs) could make excellent biomarkers of disease and different physiologic states, including pregnancy status. We tested the hypothesis that circulating extracellular vesicle-derived miRNAs might differentiate the pregnancy status of cows that had maintained pregnancy to Day 30 from non-pregnant cows or from those that exhibited embryonic mortality between Days 17 and 30 of gestation. Cows were randomly assigned for artificial insemination with fertile semen (n = 36) or dead semen (n = 8; control group) on Day 0 (day of estrus). Blood was collected from all animals on Day 0 and on Days 17 and 24 after artificial insemination. Cows receiving live sperm were retrospectively classified as pregnant on Day 30 (n = 17) or exhibiting embryonic mortality between Days 17 and 30 (n = 19). Extracellular vesicles from Day 17 and 24 samples were isolated from serum using ultra-centrifugation, and their presence was confirmed by nanoparticle tracking and Western blot analyses (for CD81) prior to RNA extraction. MicroRNA sequencing was performed on pregnant, embryonic-mortality, and control cows (n = 4 per day), for a total of 24 independent reactions. In total, 214 miRNAs were identified in serum, 40 of which were novel. Based on differential abundance parameters, we identified 32 differentially abundant loci, representing 27 differentially abundant mature miRNA. At Days 17 and 24, specific miRNAs (e.g., miR-25, -16b, and -3596) were identified that differentiated the pregnancy status. In summary, we identified several circulating extracellular vesicles derived miRNAs that differ in abundance between embryonic mortality and pregnant cows.

Relaxin as a hormonal aid to evaluate pregnancy and pregnancy loss in bottlenose dolphins (Tursiops truncatus).

This study was conducted to critically evaluate weekly and monthly circulating concentrations of immunoreactive relaxin throughout pregnancies that resulted in live births, stillbirths, and abortions in aquarium-based bottlenose dolphins. A relaxin RIA was used to analyze serum collected during 74 pregnancies involving 41 dolphins and 8 estrous cycles as well as 8 non-pregnant dolphins. Pregnancies resulted in live births (n=60), stillbirths (n=7), or abortions (n=7). Relative to parturition (Month 0), monthly changes (P<0.0001) in relaxin was indicated by relatively low concentrations during early pregnancy (Months -12 to -9) which subsequently increased (P<0.05) during mid- (Months -8 to -5) to late (Months -4 to -1) pregnancy; relaxin was highest (P<0.05) at the time of parturition. Post-parturition (Month 1), concentrations decreased (P<0.05). During the first 4weeks post-ovulation, relaxin concentrations were not different between pregnant and non-pregnant dolphins (status-by-week interaction, P=0.59). Status-by-month interaction (P<0.0002) involving different pregnancy outcomes was due, impart, to an increase in relaxin during early pregnancy (P<0.05) that was comparable among dolphins with live births, stillbirths, and abortions except concentrations were lower (P<0.05; 52%) at mid-pregnancy in association with pregnancy loss. Thereafter, concentrations increased (P<0.05) during late pregnancy in dolphins with stillbirths but not in dolphins with abortions. In conclusion, this study provided new information on the pregnancy-specific nature of relaxin, critical evaluation of the fundamental characteristics of relaxin during pregnancy and pregnancy loss, and clarification on the strengths and limitations of relaxin as a diagnostic aid to determine pregnancy status and assess maternal-fetal health in bottlenose dolphins.

The concentration-dependent effect of progesterone on follicle growth in the mouse ovary.

Follicle growth in the mammalian ovary is coordinately controlled by multiple factors to sustain periodic ovulation. In this study, we investigated the role of progesterone on follicle growth in the mouse ovary. As the concentration of progesterone changes during the estrus cycle, we cultured the sliced mouse ovary in a medium containing 10 ng/ml, 100 ng/ml, and 1 µg/ml progesterone. Progesterone promoted the growth of primordial to primary follicles at 100 ng/ml, while it suppressed the growth of secondary follicles at 1 µg/ml. Follicles at other developmental stages in the cultured ovary were unaffected with different concentrations of progesterone. The number of ovulated oocytes increased in the medium containing 100 ng/ml progesterone but decreased in the presence of 1 µg/ml progesterone. Follicles expressed two types of progesterone receptors, progesterone receptor (PGR) and PGR membrane component 1 (PGRMC1). While PGR shows transient expression on granulosa cells of Graafian follicles, PGRMC1 expresses in granulosa cells of developing follicles. These results suggest that progesterone controls the growth of developing follicles through PGRMC1. Our study shows that the effect of progesterone on ovulation and follicle growth in mouse ovary is dependent on the concentration of progesterone and the follicle stage.

High urea and pregnancy or conception in dairy cows: A meta-analysis to define the appropriate urea threshold.

Dietary proteins play an important role in reproduction, and increased dietary crude proteins, increased degradability of dietary proteins, and elevated blood or milk urea have been associated with decreased conception and pregnancy in many studies. The aim of this work was to provide a meta-analysis on the relationship between high milk or blood urea and pregnancy or conception, with a focus on defining the appropriate urea threshold associated with this issue. The meta-analysis included 61 different models from 21 papers. The thresholds of urea tested in the various models were built by steps of 1 mM urea. This constructed variable reduced heterogeneity by 61% in the meta-regression. The meta-analysis showed 43% lower odds of pregnancy or conception (odds ratio = 0.57; 95% confidence interval = 0.45-0.73) in cases where urea was ≥7.0 mM in the blood (plasma urea nitrogen = 19.3 mg/dL) or where urea was ≥420 mg/L in the milk compared with where urea values were lower. This threshold is the most suitable with regard to pregnancy or conception success, even if a threshold of 6.5 mM cannot be excluded with certainty. The results also highlighted the possibility of a stronger association between high urea concentrations and pregnancy or conception when high nitrogen exposure occurs before artificial insemination compared with after artificial insemination, but this possibility needs to be further studied. Whether the present results also apply to extensively pasture-based countries remains to be determined.

Serum estradiol and progesterone profiles during estrus, pseudopregnancy, and active gestation in Steller sea lions.

While the proximate driver behind the decline of the Western stock of Steller sea lions (Eumetopias jubatus, >80% since 1970s) is likely multifactorial, the population reduction may have been powered by a decrease in fecundity. A harvest of Steller sea lions in the 1970s and 80s revealed a 30% reduction in the proportion of pregnant females from early (October-November) to late gestation (April-May). Identification and quantification of these reproductive failures are difficult when we lack species-specific data on endocrinology associated with discrete stages of the reproductive cycle (i.e., estrus, implantation, and gestation). We tracked changes in serum estradiol and progesterone in three adult female Steller sea lions from 2011 to 2015. In all years and most females, a discrete increase in estradiol was observed during the breeding season (June-August), indicative of estrus. Estradiol concentrations from October to May in a pregnant female compared to her corresponding values when non-pregnant did not consistently differ through gestation. An elevation in progesterone was observed in all females and all years beginning approximately in June and lasting through November. This likely results from progesterone production by the corpus luteum in both pregnant and pseudopregnant females. Serum progesterone shows promise as a diagnostic tool to identify pregnancy during months 3-5 (December-February) of the 8-month active gestation following embryonic implantation. This study provides ranges of key hormones during estrus, embryonic diapause/pseudopregnancy, and gestation in pregnant and non-pregnant females for studying reproduction in Steller sea lions.