temperatures. Due to toxicity concerns of the permeable CPAs, disaccharides (trehalose, maltose and sucrose), amino
acids (EDTA, Proline, Glycine, Glutamic acid, Aminobutyric acid, Glutaric acid, Ammonium acetate), and proteins (Bovine
Mosca et al., 2016). Considering how other insect species have successfully used Trehalose for, sperm preservation,
Threhalose was selected as a good candidate to test to see if it can increase preservation of honey bee sperm as well. The
objective of the ongoing experiments was to evaluate the effects of various concentrations of Trehalose (0.05M, 0.1M or
none at all) as viable freezing extenders that are also supplemented with 12% DMSO. The effectiveness of treatments on
extending the shelf-life of honey bee spermatozoa was evaluated based on post-thaw drone semen motility and plasma
membrane functional integrity.
MATERIALS AND METHODS
Apis mellifera anatoliaca drones were collected from the Beekeeping Development-Application and Research
Center (AGAM) colonies and semen freezing was carried out at the Laboratory of Andrology within the Faculty of
Veterinary Medicine, located in Bursa Uludag University, Turkey between May and July 2017. Mature drones that were 16
days age or older chosen from 5 different colonies that were established with drone wax foundations. The colonies used in
this experiment were managed using standard beekeeping practices and were only treated for Varroa mite infestations
using amitraz strips.
Catalase, DMSO (D2650), Trehalose and Amoxicillin were bought from Sigma Aldrich (Sigma Chemical Co., St. Louis,
MO, USA). The other chemicals (Na citrate, NaHCO3, KCl) used were purchased from Merck (Merck & Co., Inc. Germany).
Semen collection and dilution
syringe tip 1.10 (Schley Instrumental Insemination equipment, Lich, Germany) under a stereo microscope (Collins and
Donoghue, 1999). A total of 0.8 L of saline solution was drawn into a capillary, followed by 3L of semen (approximately
3-4 mature drones) for each freezing group.
The diluted and thawed sperm motility was assessed under a phase-contrast microscope, at 400 X magnification.
Observed sperm motility was scored on a scale of 0 to 5 corresponding to 0%, 20%, 40%, 60%, 80% and ≥80% amount of
Plasma membrane integrity
Sperm plasma membrane functional integrity was evaluated by the Water test (Nur et al., 2012). The 1.0 L of
diluted semen was added to 250 L of distilled water and rested for 5 min at room temperature. One drop of incubated
semen was placed on a microscope slide and the percentage of coiled tail spermatozoa was determined under a phase-
contrast microscope (400X) (Nur et al., 2012). Sperm plasma membrane integrity was also assessed at the post-thaw
stage as well.
Semen dilution and freezing
As suggested by Taylor et al. (2009) extender solutions IV (Na Citrate 2.43g, NaHCO3 0.21g, KCl 0.04g, Amoxicillin
0.03g, Catalase 200L in a volume 100ml) were used. The extender’s Ph was fixed to 8.1 and split into three groups I:
Trehalose free (control group), II: 0.05 M Trehalose and 0.1M Trehalose. Obtained semen (3L) was diluted (1 part semen
to10 parts extender solutions) with one of the freezing extender group solutions at room temperature and loaded into a
0.25ml straw (Alcay et al., 2015). The semen filled straws were cooled to 5ºC within 1 h in a cold cabinet and then were
equilibrated for 2 h. Equilibrated straws were then frozen in liquid nitrogen vapor (5cm above the level of LN2, −80ºC-120)
for 10 min and then dipped into liquid nitrogen at roughly -196C. From each group two straws were thawed at 37ºC for
30 s in a water bath which then followed with a post-thaw semen motility and plasma membrane integrity evaluation. The
procedure was repeated 5 times for each group (5x2=10straws).
The obtained data were subjected to the Kruskal Wallis –Mann Whitney U test for the motility and plasma
membrane integrity tests, comparing across the different extender solutions for cryopreservation of spermatozoa. The
SPSS statistical package (SPSS 10.0 for Windows, SPSS, Chicago, IL, U.S.A), was used for all statistical tests.
The semen motility and plasma membrane integrity as a function of Trehalose content at the two different stages were
presented in Figures 1 and 2. There were no differences among diluted semen motility between control and the treatment
Citation: Nur Z, Seven Çakmak S, Çakmak İ, Onder NT, Gokçe E, Ustuner B, Alcay S, Toker MB, Soylu MK (2020). Effects of trehalose supplementation on post-thaw
sperm quality of honey bee drones. Online J. Anim. Feed Res., 10(5): 191-196.