Meta-analysis to determine efficacy of colony-stimulating factor 2 for improving pregnancy success after embryo transfer in cattle.

Results have been inconsistent as to whether addition of colony stimulating factor 2 (CSF2) to culture medium improves embryo competence for establishment of pregnancy in cattle and humans. The purpose of the current study was to use all available experiments in cattle concerning effects of CSF2 on pregnancy success after transfer into recipient cattle. The approach was to perform a meta-analysis of all published data sets as well as data from an unpublished experiment described for the first time here. Meta-analysis failed to support the hypothesis that addition of CSF2 to embryo culture medium improves competence of bovine blastocysts to increase pregnancy or calving rates after transfer into recipient females. Thus, its general use as a culture medium additive to increase pregnancy success after embryo transfer is not recommended.

Identification of large offspring syndrome during pregnancy through ultrasonography and maternal blood transcriptome analyses.

In vitro production (IVP) of embryos in cattle can result in large/abnormal offspring syndrome (LOS/AOS) which is characterized by macrosomia. LOS can cause dystocia and lead to the death of dam and calf. Currently, no test exists to identify LOS pregnancies. We hypothesized that fetal ultrasonography and/or maternal blood markers are useful to identify LOS. Bovine fetuses were generated by artificial insemination (control) or IVP. Fetal ultrasonographies were taken on gestation D55 (D55) and fetal collections performed on D56 or D105 (gestation in cattle ≈ D280). IVP fetuses weighing ≥ 97 percentile of the control weight were considered LOS. Ultrasonography results show that the product of six D55 measurements can be used to identify extreme cases of LOS. To determine whether maternal blood can be used to identify LOS, leukocyte mRNA from 23 females was sequenced. Unsupervised hierarchical clustering grouped the transcriptomes of the two females carrying the two largest LOS fetuses. Comparison of the leukocyte transcriptomes of these two females to the transcriptome of all other females identified several misregulated transcripts on gestation D55 and D105 with LOC783838 and PCDH1 being misregulated at both time-points. Together our data suggest that LOS is identifiable during pregnancy in cattle.

Actions of DKK1 on the preimplantation bovine embryo to affect pregnancy establishment, placental function, and postnatal phenotype†.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100 ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes.

Progesterone-dependent and progesterone-independent modulation of luminal epithelial transcription to support pregnancy in cattle.

In cattle, starting 4-5 days after estrus, preimplantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early postestrus, endometrial function is regulated by sex steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. The first objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 days (D4) after estrus. The second objective was to discover luminal transcripts that predict pregnancy outcomes when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome was determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 nonpregnant). Progesterone concentration on D4 was associated positively (n = 182) and negatively (n = 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity, and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n = 22) and negatively (n = 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64%-96% and 44%-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and progesterone-independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.

Choline acts during preimplantation development of the bovine embryo to program postnatal growth and alter muscle DNA methylation.

The preimplantation period of embryonic development can be a key window for programming of postnatal development because extensive epigenetic remodeling occurs during this time. It was hypothesized that modification of one-carbon metabolism of the bovine embryo by addition of the methyl-donor choline to culture medium would change postnatal phenotype through epigenetic modification. Embryos produced in vitro were cultured with 1.8 mM choline chloride or control medium. Blastocysts were transferred into females and pregnancy outcomes and postnatal phenotype of the resultant calves determined. Exposure of embryos to choline increased gestation length and calf birth weight. Calves derived from choline-treated embryos were also heavier at weaning and had increased ratio of body weight to hip height than control calves. Choline altered muscle DNA methylation of calves 4 months after birth. A total of 670 of the 8149 CpG examined were differentially methylated, with the predominant effect of choline being hypomethylation. Among the genes associated with differentially methylated CpG were ribosomal RNAs and genes in AMPK, mTOR, integrin, and BEX2 canonical pathways and cellular functions involved in growth and proliferation. Results demonstrate that provision of the methyl-donor choline to the preimplantation embryo can alter its developmental program to increase gestation length, birth weight, and weaning weight and cause postnatal changes in muscle DNA methylation including those associated with genes related to anabolic processes and cellular growth. The importance of the nutritional status of the embryo with respect to one-carbon metabolism for ensuring health and well-being after birth is emphasized by these observations.

Programming of postnatal phenotype caused by exposure of cultured embryos from Brahman cattle to colony-stimulating factor 2 and serum.

Alterations in the environment of the preimplantation embryo can affect competence to establish pregnancy and phenotype of resultant calves. In this study, the bovine embryo produced in vitro was used to evaluate postnatal programming actions of the embryokine colony-stimulating factor 2 (CSF2) and serum, which is a common additive of culture media. Oocytes were collected by ovum pick up from Brahman donors and fertilized with semen from Brahman bulls. Embryos were randomly assigned to one of the three treatments: vehicle, CSF2 10 ng/mL, or 1% (v/v) serum. Treatments were added to the culture medium from day 5 to 7 after fertilization. Blastocysts were harvested on day 7 and transferred into crossbred recipients. Postnatal body growth and Longissimus dorsi muscle characteristics of the resultant calves were measured. The percent of cleaved embryos becoming blastocysts was increased by serum and, to a lesser extent, CSF2. Treatment did not affect survival after embryo transfer but gestation length was shortest for pregnancies established with serum-treated embryos. Treatment did not significantly affect postnatal body weight or growth. At 3 mo of age, CSF2 calves had lower fat content in the Longissimus dorsi muscle and less subcutaneous fat over the muscle than vehicle calves. There was a tendency for cross-sectional area of the muscle to be smaller for serum calves than vehicle calves. Results confirm the importance of the preimplantation period as a window to modulate postnatal phenotype of resultant calves. In particular, CSF2 exerted actions during the preimplantation period to program characteristics of accumulation of intramuscular and subcutaneous fat of resultant calves. The use of a low serum concentration in culture medium from day 5 to 7 of development can increase the yield of transferrable embryos without causing serious negative consequences for the offspring.

Body Condition Score and Milk Production on Conception Rate of Cows under a Small-Scale Dairy System.

Management and production characteristics impact conception rate to first service (CR1S) in small-scale dairy farms, but the impact of body condition score (BCS) and milk production levels on cows' fertility is unknown. Our objective is to determine the effect of BCS and milk production on CR1S in small-scale dairy farms of western Mexico. Logistic regression models are used to determine the effect of BCS (at calving and first service), 60-d and 305-d milk production, protein and fat production, lactation number, and days at first service on CR1S. BCS at calving does not affect CR1S in cows with three or more lactations (39.5%; p > 0.1). However, first-lactation cows with BCS < 3.0 at calving and second lactation cows with BCS ≤ 2.5 at calving have higher CR1S (63.2 and 67.9%, respectively; p < 0.1). This result is perhaps due to reduced milk production, which leads to lower metabolic stress. BCS ≤ 2.5 at calving is associated (p < 0.05) with a reduced milk yield, explaining partially the observed higher CR1S in these groups. Cows with BCS ≤ 2.5 at first service in the higher quartile of 60-d milk production (≥ 28kg/day) show lower CR1S (23.9 and 51.1%, respectively; p < 0.01). In conclusion, BCS at calving and at first service, 60-d milk production, and lactation number are factors associated with CR1S.

Risk factors associated with reproductive performance in small-scale dairy farms in Mexico.

Several studies suggest that reproductive performance in small-scale dairy farms is low reducing the farms' profitability. Therefore, identifying risk factors associated with low reproductive performance is a key step to implement an improved reproductive management program. Accordingly, the aim of the present study was to identify the main risk factors affecting the reproductive performance of cows in small-scale dairy farms. Ninety-six dairy farms were incorporated into this study, and data from 1263 lactations were collected with different events as potential risk factors. Logistic regression models were used to assess the association (odds ratio, OR) and impact (population attributable fraction, PAF) between the potential risk factors and the reproductive variables. The main risk factors associated with assisted calving were male calf and primiparous cows (OR = 1.7, PAF = 0.315 and OR = 1.5, PAF = 0.131, respectively), while for retained fetal membranes (RFM) were assisted calving and abortion (OR = 4.5, PAF = 0.440 and OR = 8.1, PAF = 0.239, respectively). The main risk factors for days to first service over 70 days in milk were low body condition score at calving (BCS ≤ 2.5) and primiparous cows (OR = 2.2, PAF = 0.285 and OR = 1.4; PAF = 0.096, respectively), while for days open over 110 days in milk were low BCS at calving (BCS ≤ 2.5) and primiparous cows (OR = 1.7, PAF = 0.213 and OR = 1.4; PAF = 0.096, respectively) The main risk factor for non-pregnant cows at first service was RFM (OR = 1.7; PAF = 0.059). In conclusion, assisted calving, male calf, BCS ≤ 2.5 and RFM were the main risk factors associated with reduced reproductive performance in small-scale dairy farms in tropical and subtropical regions of Mexico.

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.

Nutritional status influences reproductive seasonality in Creole goats: 1. Ovarian activity during seasonal reproductive transitions.

The objective was to determine the effect of body energy stores, evaluated by a body mass index (BMI), and food intake (FI), on the length of the anovulatory period and ovarian activity during the seasonal reproductive transitions in Creole goats. Non-pregnant, non-lactating Creole goats (n=28) were fed to induce two different BMI conditions: Greater (GBMI; n=15), and Lesser (LBMI; n=13). Each BMI group was divided into two sub-groups, which were either feed restricted (FR) or non-feed restricted (NFR). Goats in the NFR groups received a diet containing 100% of the daily maintenance requirements (basal diet), while restricted goats were subjected to alternated periods, receiving 100% (11d) and 60% (10d) of the basal diet, during the entire experimental period. The experiment started after does were treated to synchronize time of estrus. Serum progesterone was determined in samples obtained twice a week, and used as a criterion for determining ovulations. During the transition to the anovulatory period three transrectal ovarian ultrasonographic scans were performed in a sub-group of 12 goats (n=3 for each treatment combination). The diameter of the largest follicle (LFD) and the total number of antral follicles >or=2mm (TAF) were recorded. Ultrasonographic ovarian scans were performed at 21, 42 and 63 days after the beginning of the experiment, concurrently with the end of each feed restriction period. The variables of response associated with ovulation were not influenced by BMI or BMIxFI interaction. However, FI influenced length of anovulatory season, as the anovulatory period was 30d longer (P<0.05) in the FR group as compared with the NFR group. Independently of treatments, TAF and LFD decreased from the first to the third ultrasonographic ovarian scan (13.2, 10.8 and 4.4 follicles; 3.7, 2.7 and 2.3mm). Nevertheless, in PER 1 the number of TAF was greater (P<0.05) in the FR as compared with NFR group and the GBMI group had a larger LFD (P<0.05) as compared to the LBMI group. It is concluded, that temporal restriction in feed intake could affect the time of cessation and initiation of ovulations during the periods of transition to seasonal anestrus and return to estrous activity, and increase the length of the anovulatory period. In addition, ovarian follicular development during transition into the anovulatory period is differentially influenced by food intake and the status of body energy stores.