74 Follicular fluid anti-Müllerian hormone concentration predicts juvenile ovine in vitro embryo development
Authors: Seccafien J. E. , Kelly J. M. , McGrice H. , Kleemann D. O. , Kind K. L. , van Wettere W. H. E. J. 
Published: December 2018

Currently, the commercial viability of assisted reproductive embryo technologies within the Australian livestock industry is restricted by individual variability in response to treatment protocols as well as oocyte developmental competence. The majority of losses come from embryo wastage, resulting from poor developmental competence during in vitro embryo production. Follicular fluid is readily available when oocytes are collected for in vitro embryo production from juvenile or mature ewes, making it an appropriate target for analysis of phenotypic markers of oocyte developmental competence. Plasma anti-Müllerian hormone (AMH) is correlated with pregnancy losses, oocyte recovery, and blastocyst development in sheep and cattle and is an indicator for donors that respond best to gonadotrophin stimulation protocols in sheep, cattle, and goats. The aim of the current work was to determine the relationship between follicular fluid AMH and in vitro embryo production outcomes in sheep. Briefly, pairs of ovaries from 38 abattoir-derived lambs were collected individually and transferred to the laboratory. Ovaries were aspirated for in vitro embryo production following previously described methods (Walker et al. 1996 Biol. Reprod. 55, 703-708) and follicles counted. Aspirated oocytes from each of the 38 individual lamb’s pair of ovaries were pooled [n=4.11±0.53 cumulus-oocyte complexes (COC) matured/lamb; total COC matured=156], and remained as such during maturation, fertilisation, and culture. The remaining follicular fluid was centrifuged for 10min at 3000 rpm to remove excess cells and frozen at −20°C. The AMH was measured in follicular fluid by a human AMH Gen II ELISA kit validated for ovine samples (A79766, Beckman Coulter, Brea, CA, USA). Correlations between follicular fluid AMH levels and oocyte maturation and blastocyst development were determined using simple linear regression. Animals were divided into groups based on AMH levels [low (0.5-10.8ng mL−1), medium (10.81-17.89ng mL−1), or high (17.9-19.25ng mL−1)], with an unbalanced ANOVA used to determine group effects on oocyte maturation and blastocyst development (GenStat 18th edition, VSN International, Hemel Hempstead, UK). Follicular fluid AMH was positively correlated (P&lt;0.05) with the number of follicles greater than 2mm (r2=0.120) and the proportion of COC cleaved from recovered oocytes (r2=0.134). The number of COC matured per lamb was greater for those with high and medium versus low AMH (5.6±0.97 and 4.4±0.72 versus 2.1±0.97 COC/lamb). Animals with high AMH produced more blastocysts than those with medium or low AMH, when expressed as a proportion of COC recovered (P&lt;0.002) or cleaved (P&lt;0.009) oocytes. High AMH was also correlated with a greater number of expanded blastocysts produced from cleaved oocytes (P&lt;0.042). The current data support previous evidence that AMH levels positively correlate to higher antral follicle counts. The correlation between AMH and components of oocyte developmental competence suggests intrafollicular AMH may indicate the best oocytes to use for an in vitro embryo production system.