Effects of the addition of flower honey and pine honey to extenders on spermatological characteristics in ram semen

Abstract

In the current study, the effectiveness of flower and pine honey added to the semen extender at dif- ferent proportions in freezing ram semen was investigated. Semen samples were collected from 5 Kivircik rams by electroejaculation method. Semen samples were pooled and divided into five equal fractions to form the study groups (FH1.0: 1.0% flower honey (v/v); FH2.5:2.5% flower honey (v/v); PH1.0: 1.0% pine honey (v/v); PH2.5: 2.5% pine honey (v/v); control: no honey addition). The semen samples were diluted with Tris-egg yolk-based semen extenders containing flower and pine honey at different rates. After cooling to 5 degrees C and equilibration at the same temperature for 1 hour, samples were frozen in liquid nitrogen vapor and stored in liquid nitrogen. During the post-equilibration period, total motility, progressive motility, velocity, and kinetic movement parameters were determined by computer-assisted sperm analysis (CASA) method. Acrosomal and total morphological damage rates were determined by phase contrast microscopy. After thawing, CASA and morphological damage assessments were repeated, sperm viability, mitochon- drial activity, plasma membrane integrity and acrosomal integrity were also examined by flow cytometry. The study was repeated ten times. Comparing with the control group, morphological damage rates were lower in all groups containing honey after equil- ibration (p < 0.05). On the other hand, 1.0% (v/v) flower honey added to the semen extender reduced the rate of acrosomal damage (phase-contrastmicroscopy examination) and damaged membrane ratio (florescent microscopy ex- amination) (p < 0.05). Meantime, 2.5% (v/v) flower honey increased the rate of viable spermatozoa (p < 0.05), the rate of spermatozoa with high mitochondrial activity (p < 0.05) and successfully preserved plasma membrane integrity (florescent microscopy examination) after thawing (p < 0.05). It is concluded at the end of the study that, addition of flower honey to extender could be used in cryopreservation of ram semen. Especially the adding of flower honey at the rate of 2.5% (v/v) is recommended for freezing ram semen.