Application of Plant-Based Dyes for Acrosomal Cap Visualization in Cauda Epididymal Spermatozoa of Bali Cattle

The cauda epididymis contains mature spermatozoa capable of fertilization, with acrosomal integrity being a crucial factor in their function. Damage to the acrosome impairs the viability and fertilizing ability of spermatozoa. Conventional staining methods, such as Giemsa or fluorescence microscopy, are effective but less practical in low-resource settings. The present study aimed to evaluate damage to the acrosomal cap of cauda epididymal spermatozoa in Bali cattle and to assess the integrity of the acrosomal cap using Giemsa and natural dyes such as turmeric, beetroot, and areca nut extract. The present study utilized cauda epididymal spermatozoa from Bali cattle aged 3-4 years and weighing 316-412 kg. The study employed a completely randomized design with four treatments, each replicated 20 times. The treatments were Giemsa stain 100% (Control, P0), 10% Giemsa + 90% turmeric extract (P1), 10% Giemsa + 90% beetroot extract (P2), and 10% Giemsa + 90% areca nut extract (P3). Using Giemsa and natural dyes resulted in different colors of spermatozoa acrosomes. The P0 preparations exhibited a dark purple coloration, P1 appeared yellowish, corresponding to the turmeric pigment, and P3 appeared bluish, while P2 demonstrated no detectable staining on the slides. The acrosomal caps of spermatozoa in P0 were dark purple, those in P1 were yellowish, and those in P2 and P3 were unstained (Transparent or faint). The average acrosomal cap integrity was the highest in P0, followed in order by P1, P2, and P3. Based on the current findings, it can be concluded that P1, P2, and P3 signified acrosomal cap integrity in cauda epididymal spermatozoa of Bali cattle. However, P1 produced the best results, showing a distinguishable color and the highest acrosomal cap integrity score (86.9 ± 2.0%). Beetroot and areca nut extracts produced weaker staining and lower acrosomal integrity values. Therefore, turmeric extract may serve as a practical, cost-effective, and environmentally friendly alternative for acrosome evaluation in veterinary reproductive laboratories, particularly in resource-limited settings.