Machine-learning methods applied to integrated transcriptomic data from bovine blastocysts and elongating conceptuses to identify genes predictive of embryonic competence.

Early pregnancy loss markedly impacts reproductive efficiency in cattle. The objectives were to model a biologically relevant gene signature predicting embryonic competence for survival after integrating transcriptomic data from blastocysts and elongating conceptuses with different developmental capacities and to validate the potential biomarkers with independent embryonic data sets through the application of machine-learning algorithms. First, two data sets from in vivo-produced blastocysts competent or not to sustain a pregnancy were integrated with a data set from long and short day-15 conceptuses. A statistical contrast determined differentially expressed genes (DEG) increasing in expression from a competent blastocyst to a long conceptus and vice versa; these were enriched for KEGG pathways related to glycolysis/gluconeogenesis and RNA processing, respectively. Next, the most discriminative DEG between blastocysts that resulted or did not in pregnancy were selected by linear discriminant analysis. These eight putative biomarker genes were validated by modeling their expression in competent or noncompetent blastocysts through Bayesian logistic regression or neural networks and predicting embryo developmental fate in four external data sets consisting of in vitro-produced blastocysts (i) competent or not, or (ii) exposed or not to detrimental conditions during culture, and elongated conceptuses (iii) of different length, or (iv) developed in the uteri of high- or subfertile heifers. Predictions for each data set were more than 85% accurate, suggesting that these genes play a key role in embryo development and pregnancy establishment. In conclusion, this study integrated transcriptomic data from seven independent experiments to identify a small set of genes capable of predicting embryonic competence for survival.

Effect of addition of ascorbate, dithiothreitol or a caspase-3 inhibitor to cryopreservation medium on post-thaw survival of bovine embryos produced in vitro.

Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 µM) and z-DEVD-fmk (0, 50, 100 or 200 µM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p < .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p < .001) levels of intracellular ROS and fewer (p < .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.

Importance of prostate androgen-regulated mucin-like protein 1 in development of the bovine blastocyst.

BACKGROUND: Prostate androgen-regulated mucin-like protein 1 (PARM1) is a pro-proliferative and anti-apoptotic glycoprotein involved in the endoplasmic reticulum (ER) stress response. A single nucleotide polymorphism in the coding region of PARM1 has been associated with competence of bovine embryos to develop to the blastocyst stage. Here we tested the importance of PARM1 for development by evaluating consequences of reducing PARM1 mRNA abundance on embryonic development and differentiation, gene expression and resistance to ER stress. RESULTS: Knockdown of PARM1 using an anti-PARM1 GapmeR did not affect competence of embryos to develop into blastocysts but decreased the number of trophectoderm (TE) cells in the blastocyst and tended to increase the number of cells in the blastocyst inner cell mass (ICM). Treatment of embryos with anti-PARM1 GapmeR affected expression of 4 and 3 of 90 genes evaluated at the compact-morula and blastocyst stage of development at days 5.5 and 7.5 after fertilization, respectively. In morulae, treatment increased expression of DAB2, INADL, and STAT3 and decreased expression of CCR2. At the blastocyst stage, knockdown of PARM1 increased expression of PECAM and TEAD4 and decreased expression of CCR7. The potential role of PARM1 in ER stress response was determined by evaluating effects of knockdown of PARM1 on development of embryos after exposure to heat shock or tunicamycin and on expression of ATF6, DDIT3 and EIF2AK3 at the compact morula and blastocyst stages. Both heat shock and tunicamycin reduced the percent of embryos becoming a blastocyst but response was unaffected by PARM1 knockdown. Similarly, there was no effect of knockdown on steady-state amounts of ATF6, DDIT3 or EIF2AK3. CONCLUSION: PARM1 participates in formation of TE and ICM cells in early embryonic development but there is no evidence for the role of PARM1 in the ER stress response.

Effect of addition of l-carnitine to media for oocyte maturation and embryo culture on development and cryotolerance of bovine embryos produced in vitro.

The objective of these experiments was to determine the effect of l-carnitine during oocyte maturation or embryo culture on embryo development and cryosurvival. For Experiments 1-3, embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). At d 7 after insemination, embryo development was assessed, and blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Post-thaw cryosurvival was determined by re-expansion and hatching rates at 24, 48 and 72 h post-thaw. In Experiment 1, COCs were matured with or without 3.03 mM l-carnitine. There was no effect of l-carnitine supplementation during maturation on embryo development or post-thaw cryosurvival. In experiment 2, presumptive zygotes were cultured in medium supplemented with or without 5% (v/v) fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5 and 3.03 mM. There was no effect of l-carnitine treatment on embryo development, but embryos treated with l-carnitine had increased (P ≤ 0.05) post-thaw re-expansion rates, irrespective of concentration. In experiment 3, presumptive zygotes were cultured with or without 0.75 mM l-carnitine from d 1 to d 4, from d 4 to d 7 or for the entire culture period. There was no effect of l-carnitine during culture on embryo development or post-thaw cryosurvival, regardless of the timing of addition. In Experiment 4, COCs were harvested by ovum pick-up from virgin dairy heifers (n = 24) and subjected to in-vitro embryo production with presumptive zygotes cultured with or without 0.75 mM l-carnitine. At d 7 after insemination, morula and blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Lactating Holstein cows (n = 102) were used as recipients and synchronized for timed embryo transfer. At d 7 after anticipated ovulation, a single embryo was thawed and transferred to the ipsilateral uterine horn of each recipient with a corpus luteum. Pregnancy was diagnosed at d 33, 44 and 72 of gestation. l-carnitine had no effect on the percentage of cows pregnant per embryo transfer (P/ET) after transfer of a frozen-thawed embryo. In conclusion, media supplementation with l-carnitine during in vitro embryo production can improve post-thaw cryotolerance as assessed in vitro but had no effect on P/ET after transfer of frozen-thawed embryos.

Embryo and cow factors affecting pregnancy per embryo transfer for multiple-service, lactating Holstein recipients.

The objective was to determine whether pregnancy success after embryo transfer (ET) during heat stress in multi-service Holstein cows depends upon characteristics of the embryo or recipient. Female embryos produced in vitro were cultured with either 0.0 (control) or 1.8 mM choline chloride and transferred fresh. Fresh embryos of undetermined breed and frozen Holstein embryos were used when experimental embryos were insufficient. Embryos were transferred 8 d after the last GnRH injection of an ovulation synchronization program. Embryo type [frozen vs. fresh, choline vs. control, unknown breed vs. (control + choline)] and characteristics of recipients (average of 190 d in milk at transfer) were evaluated. Pregnancy per ET was lower for cows receiving frozen embryos (7.0%; 3/43) than for cows receiving fresh embryos (26.7%; 32/120) but there were no differences between various types of fresh embryo. Pregnancy per ET was lower for cows diagnosed with metritis in the early postpartum period (7.1%; 2/28) than for cows without metritis (24.4%; 33/135). In conclusion, the use of frozen/thawed embryos produced in vitro and recipients which had metritis in the early postpartum period reduced the success of ET in multiple-service Holstein cows.

Thermoregulatory response of Brangus heifers to naturally occurring heat exposure on pasture.

Heat stress is a cause of major economic losses to cattle producers, especially in tropical and subtropical environments. The objectives of this study were to assess the phenotypic variability in core body temperature and sweating rate and to evaluate the effect of coat type, temperament, and BW on core body temperature and sweating rate in Brangus heifers. During August and September of 2016, 725 Brangus heifers were evaluated on pasture in four separate groups (n = 200, 189, 197, and 139). Environmental measurements of dry bulb temperature (Tdb) and relative humidity (RH) were measured every 15 min during the entire time of data collection and the temperature-humidity index (THI) was used to quantify heat-stress potential. Coat score, sweating rate, chute score, exit score, and live weight were recorded as the animals passed through the chute. Vaginal temperature was recorded every 5 min for five consecutive days. There was significant variation in vaginal temperature between heifers in the same environmental conditions (σ2u = 0.049), suggesting opportunities for selective improvements. A repeatability of 0.47 and 0.44 was estimated for sweating rate and vaginal temperature, respectively, suggesting that one measurement would be able to adequately describe the sweating capacity or ability to control the body temperature of an individual. Vaginal temperature increased as THI increased, with approximately 1 h lag time in the animal's response. Vaginal temperature (-0.047 °C, P = 0.015) and sweating rate were lower (-5.49 ± 2.12 g/(m2·h), P < 0.01) for heifers that demonstrated a calmer behavior in the chute. Animals with shorter, smoother hair coats had significantly lower vaginal temperatures when compared to animals with longer hair coats (P < 0.01). Also, heavier heifers in this study maintained lower (P < 0.0001) vaginal temperature than the lighter heifers. Our results showed that hair coat, temperament, and weight influenced vaginal temperature regulation.

Influence of corpus luteum and ovarian volume on the number and quality of bovine oocytes.

In order to evaluate whether ovarian volume, presence and diameter of the corpus luteum (CL) have effects on the number and quality of bovine recovered oocytes, 110 ovaries were obtained from the slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grades I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P < 0.05). Ovaries with CL showed greater numbers of good quality oocytes than ovaries without CL (P < 0.05). Further, presence of CL and its diameter positively influenced the probability of recovering good quality oocytes (P < 0.05). In conclusion, ovarian volume is not a good parameter itself to predict important ovarian characteristics; moreover, analysis of CL, its presence and diameter, may be a good tool to improve efficiency on in vitro embryo production programs.