Materials and Methods: A total of 15 ejaculates from three healthy stallions were collected and cryopreserved in the same environment. Each semen sample collected was divided into four equal parts and processed. All samples were analyzed before and after freezing for motility, viability, plasma membrane integrity, and morphology. Furthermore, twenty mares were inseminated using post-thawed semen.

Results: There were no differences observed among all extenders in all the parameters before freezing. Sperm cryopreserved using HF-20 showed better motility, viability, and plasma membrane integrity than Tris extender. The Tris extender showed the most inferior quality of post-thawed semen between all the extenders. HF-20, INRA Freeze®, and EquiPlus Freeze® extenders revealed the same capacity of semen preservation in vitro and in vivo.

OBJECTIVE: In this study, we report a rapid method for the residue analysis of IND and its metabolites, viz., IND-carboxylic acid, diaminotriazine, and triazine indanone in a wide range of palm oil matrices using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

METHOD: The optimized sample preparation workflows included two options: (1) acetonitrile extraction (QuEChERS workflow), followed by freezing at -80°C and (2) acetonitrile extraction, followed by cleanup through a C18 solid phase extraction (SPE) cartridge. The optimized LC runtime was 7 min. All these analytes were estimated by LC-MS/MS multiple reaction monitoring.

RESULTS: Both sample preparation methods provided similar method performance and acceptable results. The limit of quantification (LOQ) of IND, IND-carboxylic acid, and triazine indanone was 0.001 mg/kg. For diaminotriazine, the LOQ was 0.005 mg/kg. The method accuracy and precision complied with the SANTE/12682/2019 guidelines of analytical quality control.

CONCLUSIONS: The potentiality of the method lies in a high throughput analysis of IND and its metabolites in a single chromatographic run with high selectivity and sensitivity. Considering its fit-for-purpose performance, the method can be implemented in regulatory testing of IND residues in a wide range of palm oil matrices that are consumed and traded worldwide.

SUMMARY ANSWER: The inter-observer agreement among embryologists deciding whether to freeze blastocysts of marginal morphology was low and was not improved by a modified grading system.

WHAT IS KNOWN ALREADY: While previous research on inter-observer variability on the decision of which embryo to transfer from a cohort of blastocysts is good, the impact of grading variability regarding decision to freeze borderline blastocysts has not been investigated. Agreement for inner cell mass (ICM) and trophectoderm (TE) grade is only fair, factors which contribute to the grade that influences decision to freeze.

STUDY DESIGN, SIZE, DURATION: This was a prospective study involving 18 embryologists working at four different IVF clinics within a single organisation between January 2019 and July 2019.

PARTICIPANTS/MATERIALS, SETTING, METHODS: All embryologists currently practicing blastocyst grading at a multi-site organisation were invited to participate. The survey was comprised of blastocyst images in three planes and asked (i) the likelihood of freezing and (ii) whether the blastocyst would be frozen based on visual assessment. Blastocysts varied by quality and were categorised as either top (n = 20), borderline (n = 60) or non-viable/degenerate quality (n = 20). A total of 1800 freeze decisions were assessed. To assess the impact of grading criteria on inter-observer agreement for decision to freeze, the survey was taken once when the embryologists used the Gardner criteria and again 6 months after transitioning to a modified Gardner criterion with four grades for ICM and TE. The fourth grade was introduced with the aim to promote higher levels of agreement for the clinical usability decision when the blastocyst was of marginal quality.