Pregnancy-associated glycoproteins (PAGs) are abundantly expressed products of the placenta of species within the Cetartiodactyla order (even-toed ungulates). They are restricted to this order and they are particularly numerous in the Bovidae. The PAGs exhibit a range of temporal and spatial expression patterns by the placental trophoblasts and probably represent a group of related proteins that perform a range of distinct functions in the epitheliochorial and synepitheliochorial placental forms. This review presents an overview of the origins of the PAGs, a summary of PAG expression patterns, and their use as markers of pregnancy status. Speculations about their putative role(s) in pregnancy are also presented.Reproduction (2015) 149 R115-R126

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 17–30 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 17–30 (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.

The objective of this study was to examine the association between increased physical activity at the moment of timed artificial insemination (AI), detected by an automated activity monitor (AAM), and fertility outcomes. This paper also investigated factors affecting estrous expression in general. A total of 1,411 AI events from 1,040 lactating Holstein cows were recorded, averaging 1.3 ± 0.6 (±standard deviation) events per cow. Activity (measured as steps/h) was monitored continuously by a leg-mounted AAM located on the rear leg of the cow. Ovulation was synchronized by a timed AI protocol based on estradiol and progesterone. Ovarian ultrasonography was performed in all cows on d −11 (AI = d 0) and in a subset of cows on d 0 (n = 588) and d 7 (n = 819) to determine the presence of a corpus luteum and follicles. The body condition score (1 to 5 scale) was assessed on d 0 and a blood sample was collected for progesterone measurement on d 7. Using the AAM, an estrus event was determined when the relative increase (RI) in physical activity of the cow exceeded 100% of the baseline activity. The physical activity was classified as strong RI (≥300% RI), moderate RI (100-300% RI), or no estrus (<100% RI). Milk production was measured daily and averaged between d −11 and 0. Pregnancy was diagnosed at 32 and 60 d post-AI and pregnancy losses were calculated. The mean RI at estrus was 328.3 ± 132.1%. Cows with strong RI had greater pregnancy per AI than those with moderate RI and those that did not express estrus (35.1 vs. 27.3 vs. 6.2%). When including only cows that successfully ovulated after timed AI, those that displayed strong intensity RI still had greater pregnancy per AI than those with moderate intensity RI or those that did not express estrus (45.1 vs. 34.8 vs. 6.2%).Cows expressing strong RI at timed AI had greater ovulation rates compared with moderate RI and cows that did not express estrus (94.9 vs. 88.2 vs. 49.5%). Furthermore, pregnancy losses were reduced in cows with strong RI compared with cows expressing moderate RI (13.9 vs. 21.7%). Cows with a strong RI at estrus were more likely to have a corpus luteum at the beginning of the protocol and had greater concentration of progesterone 7 d post-AI. Multiparous cows expressed lower RI compared with primiparous cows. Cows with lower body condition score tended to have decreased RI at estrus. No correlation between estrous expression and pre-ovulatory follicle diameter was observed. Also, no correlation was observed between milk production at AI and RI. In conclusion, strong intensity RI of estrus events at timed AI was associated with improved ovulation rates and pregnancy per AI, and reduced pregnancy losses. These results provide further evidence that measurements of estrous expression can be used to predict fertility at the time of AI and possibly be used as a tool to assist decision making strategies of reproduction programs.

Cattle producers are limited to day 28-30 of gestation as the earliest time point for accurate pregnancy diagnosis due to the effectiveness of ultrasound and chemical based methods, including commercially available pregnancy associated glycoproteins (PAG) tests. The objective of the current studies were to determine if early gestation circulating PAG concentrations at day 24 could be used to diagnose pregnancy in dairy cattle undergoing embryo transfer. In vitro produced embryos were transferred into Holstein x Gir cows and heifers on day 7 following ovulation. Study 1 utilized only cows (n = 101) determined to be pregnant on day 24 of gestation by progesterone concentration, as well as CL and PAG presence. In study 2, animals were not predetermined to be pregnant and both heifers (n = 111) and cows (n = 242) were used. In both studies, blood was collected at day 24 for PAG analysis as well as day 31. Final pregnancy confirmation occurred on day 60 via transrectal ultrasonography. Serum PAG concentrations were quantified using an in house PAG ELISA. Following timed embryo transfer (TET) in study 1, of the 101 cows diagnosed as pregnant on day 24, 80 cows were identified as still pregnant on day 31 of gestation (77%). Study 2 had a pregnancy rate at day 31 of 33.7% of total embryos transferred. Mean circulating PAG concentration at day 24 differed (P < 0.001) between animals diagnosed pregnant and non-pregnant at day 31 in both studies (study 1, 2.964 ± 0.262 ng/mL vs 0.946 ± 0.168 ng/mL and study 2, 1.962 ± 0.261 ng/mL vs 0.731 ± 0.109 ng/mL). Concentration of PAG between pregnant and non-pregnant cows in study 1 and 2 was significant, however, pregnant heifers in study 2 (1.562 ± 0.266 ng/mL) had concentration of PAGs that only had a tendency to differ compared to non-pregnant heifers (non-pregnant, 0.799 ± 0.290 ng/mL; P = 0.0669). Only animals that were pregnant at day 31 were analyzed in late embryo mortality analysis (heifers, n = 54; cows, n = 159), defined as pregnancy loss between day 31 and 60. Between day 31 and 60, 39 (12 in study 1 and 28 in study 2) animals experienced late embryo mortality. Circulating concentrations of PAG were not significantly different (P > 0.05), in either study, at day 24 of gestation in animals that maintained pregnancy until day 60 compared to animals that lost pregnancy between day 31 and 60 (late embryo mortality, LEM). In summary, early gestation circulating PAG concentration may have application in diagnosing pregnancy at day 24 of gestation and more work is needed to determine the potential of early gestation PAGs in predicting embryo loss in dairy.

Pregnancy establishment, followed by birth of live offspring, is essential to all mammals. The biological processes leading up to pregnancy establishment, maintenance, and birth are complex and dependent on the coordinated timing of a series of events at the molecular, cellular, and physiological level. The ability to ovulate a competent oocyte, which is capable of undergoing fertilization, is only the initial step in achieving a successful pregnancy. Once fertilization has occurred and early embryonic development is initiated, early pregnancy detection is critical to provide proper prenatal care (humans) or appropriate management (domestic livestock). However, the simple presence of an embryo, early in gestation, does not guarantee the birth of a live offspring. Pregnancy loss (embryonic mortality, spontaneous abortions, etc.) has been well documented in all mammals, especially in humans and domestic livestock species, and is a major cause of reproductive loss. It has been estimated that only about 25-30% of all fertilized oocytes in humans result in birth of a live offspring; however, identifying the embryos that will not survive to parturition has not been an easy task. Therefore, investigators have focused the identification of products in maternal circulation that permit the detection of an embryo and assessment of its well-being. This review will focus on the advances in predicting embryonic presence and viability, in vivo.

Poor utilization of amino acids contributes to losses of milk protein yield in dairy cows exposed to heat stress (HS). Our objective was to test the effect of essential amino acids on milk production in lactating dairy cows exposed to short-term HS conditions. To achieve this objective, 12 multiparous, lactating Holstein cows were assigned to two environments (thermoneutral (THN) or HS) from days 1 to 14 in a split-plot type cross-over design. All cows received 0 g/day of essential amino acids from days 1 to 7 (negative control (NC)) followed by an intravenous infusion of l-methionine (12 g/day), l-lysine (21 g/day), l-leucine (35 g/day), l-isoleucine (15 g/day) and l-valine (15 g/day, methionine, lysine and branched-chain amino acids (ML+BCAA)) from days 8 to 14. The basal diet was composed of ryegrass silage and hay, and a concentrate mix. This diet supplied 44 g of methionine, 125 g of lysine, 167 g of leucine, 98 g of isoleucine and 109 g of valine per day to the small intestine of THN cows. Temperature-humidity index was maintained below 66 for the THN environment, whereas the index was maintained above 68, peaking at 76, for 14 continuous h/day for the HS environment. Heat stress conditioning increased the udder temperature from 37.0°C to 39.6°C. Cows that received the ML+BCAA treatment had greater p.m. rectal and vaginal temperatures (0.50°C and 0.40°C, respectively), and respiration rate (8 breaths/min) compared with those on the NC treatment and exposed to a HS environment. However, neither NC nor ML+BCAA affected rectal or vaginal temperatures and respiration rates in the THN environment. Compared with THN, the HS environment reduced dry matter intake (1.48 kg/day), milk yield (2.82 kg/day) and milk protein yield (0.11 kg/day). However, compared with NC, the ML+BCAA treatment increased milk protein percent by 0.07 points. For the THN environment, the ML+BCAA treatment increased concentrations of milk urea nitrogen. For the HS environment, the ML+BCAA treatment decreased plasma concentrations of arginine, ornithine and citrulline; however, differences were not observed for the THN environment. In summary, HS elicited expected changes in production; however, infusions of ML+BCAA failed to increase milk protein yield. Lower dry matter intake and greater heat load in response to ML+BCAA contributed to the lack of response in milk production in HS cows. The ML+BCAA treatment may have reduced the breakdown of muscle protein in heat-stressed cows.

The concentration of circulating pregnancy associated glycoproteins (PAGs) early in pregnancy may serve as markers to predict late embryonic mortality or fetal mortality in cattle. In this study, pregnancies were established in dairy cows, by either fixed-time AI (FTAI) or fixed-time embryo transfer (FTET) with in vitro produced embryos. Circulating PAGs were measured with different combinations of antibodies in either a laboratory-based ELISA or a commercial ELISA. For the in-house ELISA, three monoclonal 'trapping' antibodies (A6, J2, and L4) and two polyclonal 'detection' antisera (antibodies F2 or 45) were used to quantify PAGs in serum from the same cows. The different assays were identified as follows: 'Mix-45' (A6, J2, and L4 with 45), 'Mix-F2' (A6, J2, and L4 with F2), and 'L4-F2': (L4 with F2); the commercial assay was from IDEXX. Ovulation was synchronized and FTAI or FTET was performed on day 0 or 7, respectively. Ultrasound-based diagnosis of pregnancy and serum collections occurred on day 30. The proportion of cows that subsequently experienced pregnancy loss between days 30 and 60 was 23% (43 of 183) and 16% (21 of 131) for the FTAI or FTET groups, respectively. In the FTAI group, mean serum concentration of PAGs detected with Mix-45 was higher in cows that maintained pregnancy (9.2 ± 0.4 ng/ml; mean ± SEM) compared with cows that experienced pregnancy failure (3.9 ± 0.6 ng/ml) between day 30-60 (P < .001). However, there was no difference (P > .69) in circulating concentrations of PAGs between cows that experienced loss or survival between days 30 and 60 when Mix-F2 or L4-F2 were used in an in-house ELISA. Likewise, a commercial assay also did not result in measurable differences in PAG concentrations between those animals that experienced loss or survival. Following FTET, circulating concentrations of PAGs on day 30 were lower (P < .001) in cows that experienced pregnancy failure compared to cows that maintained pregnancy when the Mix-45 and the commercial assay were used, but not with the other antibody combinations. A receiver operating characteristic curve showed that only the Mix-45 antibody combination was predictive (95% accuracy) of pregnancy loss but not the other antibody combinations following FTAI. However, both Mix-45 and the commercial assay were predictive of losses following FTET. In summary, although multiple PAG assay formats have been shown to accurately detect pregnancy, the ability to predict embryo survival during early gestation appears to be antibody dependent.

This review summarizes strategies to increase overall pregnancy rates to TAI protocols, and potential areas for improvement in reproductive management of Nelore cows. Low BCS at calving and postpartum BCS loss negatively impacted percentage of pregnant females to TAI, with primiparous cows being the most sensitive to the effects of low BCS during the postpartum period. The current reproductive management recommended for replacement heifers (with TAI on day 0) consist of intravaginal progesterone device insertion from day -35 to -23, 0.6 mg i.m. injection of estradiol cypionate on day -23, insertion of another intravaginal progesterone device and 2.0 mg i.m. injection of estradiol benzoate on day -11, 12.5 mg i.m. injection of dinoprost tromethamine on day -4, intravaginal progesterone device removal and 0.6 mg i.m. injection of estradiol cypionate on day -2, and TAI on day 0. Supplementing corn for 41 days after the first TAI of the breeding season increased pregnancy to a second AI in primiparous cows, increased final proportion of pregnancy in primiparous cows, but decreased the final proportion of pregnancy in mature cows. In turn, supplementing melengestrol acetate or Ca salts of PUFA during the expected time of luteolysis was beneficial to pregnancy rates. Vaccinating cows against BoHV-1, BVDV, and Leptospira spp., particularly when both doses were administered before TAI, improved cow reproductive performance. Cow temperament has direct implications not only on reproductive efficiency of B. indicus females, but also on overall production efficiency in cow-calf system based on B. indicus cattle. Lastly, concentration of pregnancy-associated glycoproteins was driven by the ability of pregnancy maintenance and by sire used at TAI, whereas exploring this relationship might be interesting to improve sire fertility regarding late embryonic loss in Nelore females.