Single-cell transcriptional landscapes of bovine peri-implantation development.

Supporting healthy pregnancy outcomes requires a comprehensive understanding of the molecular and cellular programs of peri-implantation development, when most pregnancy failure occurs. Here, we present single-cell transcriptomes of bovine peri-implantation embryo development at day 12, 14, 16, and 18 post-fertilization. We defined the cellular composition and gene expression of embryonic disc, hypoblast, and trophoblast lineages in bovine peri-implantation embryos, and identified markers and pathway signaling that represent distinct stages of bovine peri-implantation lineages; the expression of selected markers was validated in peri-implantation embryos. Using detailed time-course transcriptomic analyses, we revealed a previously unrecognized primitive trophoblast cell lineage. We also characterized conserved and divergence peri-implantation lineage programs between bovine and other mammalian species. Finally, we established cell-cell communication signaling underlies embryonic and extraembryonic cell interaction to ensure proper early development. These data provide foundational information to discover essential biological signaling underpinning bovine peri-implantation development.

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.

Validation of pregnancy associated glycoproteins-based ELISA kits to determine early pregnancy status in lactating Nili-Ravi buffaloes.

The objective of the current study was to evaluate pregnancy-associated glycoproteins (PAGs) based enzyme-linked immunosorbent assay (ELISA) kits utilizing whole blood, serum, or milk samples for diagnosis of early pregnancy status in lactating Nili-Ravi buffaloes. Dairy buffaloes (n = 174) of mixed parity, 4-6 years of age, having mean (± standard deviation) days in milk 165 ± 87, and body condition score of 3.26 ± 0.34 were randomly enrolled in this study. Buffaloes were exposed to penile deviated bulls with 12 h interval for estrus detection during peak breeding season and eventually bred naturally at their respective standing estrus (day 0). Blood and milk samples were collected at days 24, 28, and 35 post-breeding to run a rapid visual pregnancy test® (RVPT) as a buffalo-side test or ELISA-based assay in the laboratory to detect early pregnancy status. Transrectal B-mode ultrasonography was performed to diagnose pregnancy at day 35 post-breeding and used as a gold standard to validate results of RVPT or ELISA-based tests. The RVPT is a visual readout test for pregnancy detection and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (77.9 vs. 89.7 vs. 93.3%), and accuracy (84.5 vs. 90.1 vs. 94.2%) at days 24, 28, and 35 post-breeding, respectively. The PAGs were assayed using ELISA kits in serum and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (84.2 vs. 87.7 vs. 93.9%), and accuracy (82.1 vs. 88.4 vs. 93.7%) at days 24, 28, and 35 post-breeding, respectively. Similarly, PAGs were also analysed using ELISA kits in milk samples and had sensitivity (77.6 vs 89.5 vs 95.0%), specificity (89.1 vs 91.9, vs 93.9%), and accuracy (85.1 vs 91.1 vs 94.3%) at days 24, 28, and 35 post-breeding, respectively. Overall, the Kappa values in this study exceeded 0.85 at day 35 post-breeding using RVPT or ELISA-based test kits in serum or milk samples, indicating a high level of agreement between PAGs detection method and gold standard for pregnancy diagnosis. The pregnancy outcomes based on ELISA-based PAGs detection at day ≥28 post-breeding had a high negative predictive value, indicating that the probability of incorrectly administering prostaglandins to pregnant buffaloes would be low if these tests were implemented on a commercial dairy herd. Taken together, it is concluded that PAGs-based determination of pregnancy using RVPT or ELISA in blood, serum, or milk samples can be used effectively for pregnancy diagnosis at ≥28 days post-breeding with more than 90% accuracy in Nili-Ravi buffaloes.

Vitrification improves in-vitro embryonic survival in Bos taurus embryos without increasing pregnancy rate post embryo transfer when compared to slow-freezing: A systematic meta-analysis.

Objectives were to use meta-analytic approaches to compare slow-freezing (SF) and vitrification (VF) methods of cryopreservation on in-vitro (n = 12,211) and in-vivo (n = 3473) survival of Bos taurus embryos. The literature was systematically reviewed and data from 40 manuscripts including 78 experiments, and comprising 183 treatment means, were used for the analyses. The in-vitro parameters included rates of re-expansion, hatching, and survival of blastocysts either at 24 h or 72 h post-thawing/warming and total number (TN) of embryonic cells, whereas in-vivo parameters evaluated pregnancy rate between 35 and 60 d post embryo transfer (ET). Mixed models were fitted using MIXED and GLIMMIX procedures of SAS. Additionally, classical meta-analytical statistics were also fitted using METAN and METAREG procedures of STATA. The final models included the fixed effects of methods of cryopreservation and random effects of the experiment. Rates (LSM ± SEM) of re-expansion (0.36 ± 0.07 vs. 0.48 ± 0.08), hatching (0.25 ± 0.05 vs. 0.42 ± 0.07), and survival (0.57 ± 0.09 vs. 0.76 ± 0.07) at 72 h post-thawing/warming were lower (P < 0.05) in SF than VF, respectively. The TN of embryonic cells (96.89 ± 7.15 vs. 117.83 ± 7.15) remained lower (P < 0.05) in SF than VF, however, the relative risk (RR) of pregnancy rate post ET remained similar (RR = 1.0, CI = 0.8-1.2; P > 0.05) between both methods. Collectively, VF technique has a short-term protective effect against cryodamage of preimplantation embryos, however, it might be dysregulating genes involved in pregnancy success post ET in cows.

Meta-analysis to determine effects of treatment with FSH when there is progestin-priming on in-vitro embryo production using ovum pick-up in Bos taurus cows.

Treatments with follicle stimulating hormone (FSH) to enhance ovarian follicular development before ovum pick-up (OPU) are important for improving in-vitro embryo production (IVEP) procedures in cows, however, their procedural efficacy needs to be evaluated. The objective of the present study was to use meta-analytic methods to determine the effects of FSH treatments prior to OPU when there is progestin-priming on ovarian functions and IVEP in parous Bos taurus cows (n = 243). The literature was systematically reviewed and data from eight experiments, with 23 treatment means including 448 OPU sessions, were used for analyses. All eight experiments included a group of cows in which there was no FSH treatment (CON), and treatment with FSH before OPU to conduct IVEP. Mixed models were fitted using MIXED and GLIMMIX procedures of SAS. Additionally, classical meta-analytical statistics were also fitted using METAN and METAREG procedures of STATA. Models included fixed effects of treatment (i.e., CON and FSH) and random effect of the experiment, and data were weighted using the inverse of the SEM squared to account for precision of each experiment. Number of medium sized follicles, quality of oocytes, and transferable embryos were greater (P < 0.05) in FSH-treated as compared with cows of the CON group, without indications of publication bias or heterogeneity of data. Taken together, the results from the present study lead to the recommendation for treatments with FSH prior to OPU for IVEP when there was progestin-priming imposed to obtain a large number of transferable embryos for economic sustainability of commercial embryo transfer programs in parous non-lactating Bos taurus cows.

Effect of photoperiodicity and methods of follicular wave emergence on follicle turn-over, recovery and quality of oocytes, and early in-vitro developmental competence of embryos using ovum pick-up in Nili-Ravi buffaloes: Preliminary evidence.

The objectives of the present study were to evaluate the effects of photoperiodicity, gauge (G) of ovum pick-up (OPU) needle, and two methods of follicular wave emergence on follicular turn-over, oocyte recovery (OR), quality of the oocytes (OQ), and early in-vitro developmental competence of embryos in Nili-Ravi buffaloes (n = 20). In 1st experiment, buffaloes (n = 12; 4 buffaloes/season) were randomly assigned to optimize the OPU's (n) either with 17 G or 18 G needle in one of the following seasons: 1) peak breeding season (PBS; Sep-Nov; n = 31), 2) transition breeding season (TBS; Dec-Feb; n = 32), and 3) low breeding season (LBS; Apr-June; n = 32). During 2nd experiment, buffaloes (n = 8) were enrolled randomly in a 2 × 2 cross-over design to compare the two methods of wave emergence either using follicular ablation (FA; n = 4), or synchronization protocol (CIDR-EB; n = 4) during PBS. In FA method, the ovarian follicles were aspirated (week -1), and subsequently repeated OPU's (n = 55) were performed for 7 weeks. However, in CIDR-EB synchronized buffaloes, a progesterone device (CIDR) was inserted in the anterior vagina and a single dose of estradiol benzoate (2 mg) and prostaglandin (150 µg) were administered i.m. on d 1. The CIDR was removed on d 7 and repeated OPU's (n = 56) were performed. In both experiments, a 7-d resting period was provided between each OPU session. Data on the follicular turn-over, OR, OQ, and early stages of embryonic development were analyzed with mixed models using the PROC MIXED and GLIMMIX procedures of SAS. Results revealed that the number of medium sized follicles (LSM ± SEM) and rate of OR (%) were greater (P < 0.05) during PBS (2.76 ± 0.40; 61%) and TBS (1.73 ± 0.30; 54%) as compared to LBS (0.68 ± 0.30; 31%) in buffaloes, respectively. The OR rate was also greater (P < 0.05) using 17 G as opposed to 18 G needle (62% vs. 35%), however, there was no effect (P > 0.05) of season and G of needle on OQ in buffaloes. In experiment 2, the number of small (3.50 ± 0.63 vs.2.69 ± 0.60) sized follicles was higher (P < 0.05) in FA, whereas, medium (1.13 ± 0.45 vs. 1.59 ± 0.45) sized follicles were greater (P < 0.05) in CIDR-EB synchronized buffaloes. The OR rate (67% vs. 53%), and OQ (Grade I_+_II; 15% vs. 16% and Grade III_+_IV; 85% vs. 84%), remained similar (P > 0.05) in FA and CIDR-EB treated buffaloes, respectively. Similarly, rates of in-vitro maturation (66% vs. 59%), cleavage (42% vs. 53%), 4-cell (27% vs. 32%), 8-cell staged embryos (23% vs. 25%), and morula (13% vs. 8%) did not differ (P > 0.05) between FA and CIDR-EB methods of follicular wave emergence, respectively. Taken together, it is concluded that peak or transition breeding seasons are suitable to perform OPU using 17 G needle for maximum follicular turn-over, and OR in buffaloes. However, the two fundamental methods of synchronization of wave emergence resulted in similar efficiency for OPU derived in-vitro embryo production in Nili-Ravi buffaloes.