Machine learning to identify endometrial biomarkers predictive of pregnancy success following artificial insemination in dairy cows†.

The objective was to identify a set of genes whose transcript abundance is predictive of a cow's ability to become pregnant following artificial insemination. Endometrial epithelial cells from the uterine body were collected for RNA sequencing using the cytobrush method from 193 first-service Holstein cows at estrus prior to artificial insemination (day 0). A group of 253 first-service cows not used for cytobrush collection were controls. There was no effect of cytobrush collection on pregnancy outcomes at day 30 or 70 or on pregnancy loss between days 30 and 70. There were 2 upregulated and 214 downregulated genes (false discovery rate < 0.05, absolute fold change >2-fold) for cows pregnant at day 30 versus those that were not pregnant. Functional terms overrepresented in the downregulated genes included those related to immune and inflammatory responses. Machine learning for fertility biomarkers with the R package BORUTA resulted in identification of 57 biomarkers that predicted pregnancy outcome at day 30 with an average accuracy of 77%. Thus, machine learning can identify predictive biomarkers of pregnancy in endometrium with high accuracy. Moreover, sampling of endometrial epithelium using the cytobrush can help understand functional characteristics of the endometrium at artificial insemination without compromising cow fertility. Functional characteristics of the genes comprising the set of biomarkers is indicative that a major determinant of cow fertility, at least for first insemination after calving, is immune status of the uterus, which, in turn, is likely to reflect the previous history of uterine disease.

Transcriptomic profiling of the bovine endosalpinx and endometrium to identify putative embryokines.

The objectives of the present study were to characterize the expression of genes encoding for cell signaling ligands in the bovine endosalpinx and endometrium and analyze spatial changes in gene expression. RNA sequencing was performed for the endosalpinx from the ampulla of the oviduct and endometrium from the upper and middle uterine horn and uterine body at day 2 after ovulation from ipsilateral and contralateral sides relative to the ovulatory ovary. Of the 17,827 unique mRNA transcripts mapped, 2,072 were affected by cranial-caudal position in the reproductive tract and 818 were affected by side (false discovery rate < 0.05). There were 334 genes encoding for cell signaling ligands, with 128 genes having greater than two transcripts per million on average. A total of 81 cell signaling ligand genes were affected by position and 24 were affected by side. A data set of the transcriptome of two to four cell embryos was used to identify cell signaling ligand genes that were highly expressed in the ampulla for which there was high expression of the receptor in the embryo. The most expressed ligand-receptor pairs were PSAP/SORT1, MIF/CXCR4, GPI/AMFR, and KITLG/KIT. These cell signaling ligands, as well as others whose gene is expressed in the endosalpinx and endometrium, may influence early embryonic development. Spatial changes throughout the reproductive tract highlight the distinctive expression profile of the oviduct versus the endometrium, including a set of the identified genes encoding for cell signaling ligands, and highlight the local influence of the ovary. The results also show the continuity of expression for large numbers of genes in the reproductive tract.NEW & NOTEWORTHY Examination of the transcriptome of the endosalpinx and endometrium revealed the degree to which gene expression in the reproductive tract varies spatially. The expression of genes encoding cell signaling molecules that could potentially regulate embryonic development was also identified.

A low concentration of choline chloride alters the developmental program of the bovine preimplantation embryo.

Choline is a known developmental programming agent of the bovine preimplantation embryo. Culture of the embryo with 1.8 mmol/L choline, a concentration much higher than in blood, alters development to cause increased weaning weight and other changes during the postnatal period. It was hypothesized here that choline exerts similar effects on the developmental program of the embryo when added at concentrations similar to those in peripheral blood (i.e., 4 mol/L). Oocytes were collected via ovum pick up and embryos were produced in vitro. Embryos were cultured until day 7 after fertilization in medium with 4 mol/L choline chloride, or, as a vehicle control, with an additional 4 mol/L sodium chloride. Blastocysts were transferred into recipients and pregnancy was diagnosed at approximately 28 d of gestation. Subsequent calves (n=37 for vehicle and n=35 for choline) were weighed at birth and at weaning. Addition of choline to culture medium did not affect the proportion of embryos that became blastocysts or the proportion of transferred blastocysts that produced a pregnancy. Birth weight was unaffected by treatment but calves derived from choline-treated embryos were heavier at time of weaning and gained more per day from birth until weaning than calves derived from embryos treated with vehicle. Results demonstrate that choline can act on the preimplantation embryo at a physiologically-relevant concentration to alter postnatal phenotype. Observations are further evidence for the importance of the first days of embryonic development for the phenotype of the resulting calf.

Consequences of gene editing of PRLR on thermotolerance, growth, and male reproduction in cattle.

Global warming is a major challenge to the sustainable and humane production of food because of the increased risk of livestock to heat stress. Here, the example of the prolactin receptor (PRLR) gene is used to demonstrate how gene editing can increase the resistance of cattle to heat stress by the introduction of mutations conferring thermotolerance. Several cattle populations in South and Central America possess natural mutations in PRLR that result in affected animals having short hair and being thermotolerant. CRISPR/Cas9 technology was used to introduce variants of PRLR in two thermosensitive breeds of cattle - Angus and Jersey. Gene-edited animals exhibited superior ability to regulate vaginal temperature (heifers) and rectal temperature (bulls) compared to animals that were not gene-edited. Moreover, gene-edited animals exhibited superior growth characteristics and had larger scrotal circumference. There was no evidence for deleterious effects of the mutation on carcass characteristics or male reproductive function. These results indicate the potential for reducing heat stress in relevant environments to enhance cattle productivity.

Identification of quantitative trait loci and associated candidate genes for pregnancy success in Angus-Brahman crossbred heifers.

BACKGROUND: In beef cattle, more than 50% of the energy input to produce a unit of beef is consumed by the female that produced the calf. Development of genomic tools to identify females with high genetic merit for reproductive function could increase the profitability and sustainability of beef production. RESULTS: Genome-wide association studies (GWAS) were performed using a single-step genomic best linear unbiased prediction approach on pregnancy outcome traits from a population of Angus-Brahman crossbred heifers. Furthermore, a validation GWAS was performed using data from another farm. Heifers were genotyped with the Bovine GGP F250 array that contains 221,077 SNPs. In the discovery population, heifers were bred in winter breeding seasons involving a single round of timed artificial insemination (AI) followed by natural mating for 3 months. Two phenotypes were analyzed: pregnancy outcome to first-service AI (PAI; n = 1,481) and pregnancy status at the end of the breeding season (PEBS; n = 1,725). The heritability was estimated as 0.149 and 0.122 for PAI and PEBS, respectively. In the PAI model, one quantitative trait locus (QTL), located between 52.3 and 52.5 Mb on BTA7, explained about 3% of the genetic variation, in a region containing a cluster of γ-protocadherin genes and SLC25A2. Other QTLs explaining between 0.5% and 1% of the genetic variation were found on BTA12 and 25. In the PEBS model, a large QTL on BTA7 was synonymous with the QTL for PAI, with minor QTLs located on BTA5, 9, 10, 11, 19, and 20. The validation population for pregnancy status at the end of the breeding season were Angus-Brahman crossbred heifers bred by natural mating. In concordance with the discovery population, the large QTL on BTA7 and QTLs on BTA10 and 12 were identified. CONCLUSIONS: In summary, QTLs and candidate SNPs identified were associated with pregnancy outcomes in beef heifers, including a large QTL associated with a group of protocadherin genes. Confirmation of these associations with larger populations could lead to the development of genomic predictions of reproductive function in beef cattle. Moreover, additional research is warranted to study the function of candidate genes associated with QTLs.