Following mating, only a subset of spermatozoa begin an extensive migration toward their final objective—the fertilization of the oocyte. Within the oviduct, these sperm are temporarily retained, attaching to the upper membrane of epithelial cells, forming a “sperm nest.” This reservoir performs several crucial tasks: maintaining sperm contact with epithelial cells, filtering viable and structurally sound spermatozoa, and ultimately prolonging their functional lifespan. The present work investigated the fertilization competence of sperm adhered to cellular aggregates obtained from three oviductal sections (infundibulum, ampulla, and isthmus). Additionally, it examined how heparin and/or progesterone (P4) influence the in vitro fertilization efficiency of sperm preincubated with isthmic aggregates. In vitro fertilization was defined by the cleavage of an oocyte into two or more blastomeres. The sperm attached to isthmic aggregates showed the highest fertilization percentage (48.09%) compared with those associated with infundibular (36.90%) or ampullary (37.61%) tissue. Moreover, treating mature COCs with heparin (40 μg/mL) combined with P4 (80 nM) produced a joint stimulatory effect, raising the in-vitro fertilization rate of sperm attached to isthmic aggregates to 63.33%, in contrast with 42.61% in the absence of cell aggregates. Overall, sperm interaction with isthmic cell clusters enhances bovine sperm fertilizing efficiency, and the joint presence of heparin and progesterone further potentiates this effect.
