Abstract

Reliable preimplantation embryo sexing is relevant in cattle breeding; however, PCR-based Y-marker detection may be sensitive to limited DNA input and sample-type-specific properties of embryo-derived material. In this observational study, we examined assay-output patterns in a low-input multiplex PCR workflow targeting a male-specific Y-chromosome marker (MSY) together with an autosomal internal control targeting cyclin-dependent kinase 1 (CDK1). A total of 141 bovine embryo-derived samples were analyzed across three sample categories: whole embryos, cleavage-stage blastomere biopsies, and blastocyst-stage trophectoderm biopsies. Assay evaluability differed across sample categories, being highest in blastocyst-stage biopsy samples (90.6%, 29/32), intermediate in whole embryos (82.9%, 58/70), and lowest in blastomere-biopsy samples (64.1%, 25/39). Among technically evaluable CDK1-positive samples, apparent MSY-positive classification also differed across categories, with rates of 55.2% (32/58) in whole embryos, 52.0% (13/25) in blastomere-biopsy samples, and 6.9% (2/29) in blastocyst-stage biopsy samples. In a same-lysate repeat-amplification subset, overall evaluability concordance was 92.5% and overall apparent MSY-based classification concordance was 88.2%, although subgroup sizes were limited. Because no independent reference method for true embryo sex was available and sample category was structurally confounded with developmental stage, semen type, sire, and partly PCR-date structure, the findings should be interpreted as assay-output observations rather than measures of biological sex ratio or diagnostic sexing accuracy. Overall, embryo-derived sample categories showed differing assay evaluability and apparent MSY-classification patterns within this low-input PCR setting, supporting the need for sample-type-specific validation when low-cell embryo-derived samples are used.

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