2020 SCIENCELINE

O nline J ournal of A nimal and F eed R esearch

Volume 10, Issue 5: 191-196; September 25, 2020

EFFECTS OF TREHALOSE SUPPLEMENTATION ON POST-

THAW SPERM QUALITY OF HONEY BEE DRONES

Zekariya NUR¹, Selvinar SEVEN ÇAKMAK², İbrahim ÇAKMAK³, Nail Tekin ONDER¹, Elif GOKÇE⁴, Burcu

USTUNER¹, Selim ALCAY¹, Mehmet Berk TOKER¹, Mustafa Kemal SOYLU¹

¹Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Bursa Uludag University, 16059, Turkey

²Department of Biology, Faculty of Science, Ankara, Turkey

³Beekeeping Development-Application and Research Center, Department of Animal Science, Faculty of Agriculture, Bursa Uludag University, 16059, Turkey

⁴Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Tekirdag Namık Kemal University, 59030, Turkey

^Email: icakmak@uludag.edu.tr;

: 0000-0002-8000-5770

^Supporting Information (This research had been funded by TÜBITAK (Project number: 205O586)

ABSTRACT: Sperm cryopreservation has led to an increase in widespread use and has it made it more practical

to use artificial insemination not only for domestic animals but also for non-mammalian species and humans.

Dimethyl Sulfoxide (DMSO) was the most frequently used cryoprotectant by protecting honey bee drone semen

when freezing it. The objective of this study was to determine the effects of Trehalose (0.05M, 0.1M or none at

all) on extending the viability of semen with 12% DMSO that was based on sperm motility and plasma

membrane functional integrity of frozen drone semen. Three different freezing extender solutions were

designated as follows; the 0.05M Trehalose, 0.1M Trehalose and Trehalose free (control group). Semen motility

and plasma membrane functional integrity were evaluated under phase-contrast microscopy (400X). We found

that in control group, DMSO is a critical substance in freezing extender and supports post-thaw sperm motility

(53%) and plasma membrane functional integrity (79%) to some extent. Addition of 0.05M Trehalose to the

extender leads to a small recovery of post-thaw motility (55%) and plasma membrane integrity (89%), but when

Trehalose is added at 0.1M concentration, this led to significantly better post-thaw motility (62%) and plasma

membrane integrity (91%). In conclusion, the freeze-thaw process is detrimental to post-thaw drone semen

viability. The addition of 0.1 or 0.05M Trehalose to the freezing media containing 12% DMSO has been seen

better post-thaw cell motility and plasma membrane integrity of spermatozoa.

Keywords: Honey bee, Drone, Semen, Cryoprotectant, DMSO, Trehalose

INTRODUCTION

Honey bee contribution in pollination of crops (Apis mellifera L.) and end products (honey, royal jelly, wax, pollen, and

propolis) are crucial for agriculture and beekeepers. Recently the variety of subspecies and populations of honey bees

have declined dramatically by virtue of man-made (habitat destruction, usage of pesticide, transport stress, management

failures and poor queen health) and natural factors (starvation, parasites, winter colony losses) (Çakmak and Çakmak,

2016; Moritz and Erler, 2016). The losses of honey bees vary from region to region, but in general recently there has been

large scale colony losses (35% to 75%) observed (Neumann and Norman, 2010). Therefore, the ability to preserve honey

bee genetic material and to artificially inseminate for breeding different honey bee genetic lines are regarded as a critical

tool for improving bee health and producing the beekeeping products mentioned above. Maintenance of genetic diversity,

protection of desirable genetic lineages, prevention of colony loss, and increasing the productivity of bee colonies are

possible if honey bee drone sperm preservation and artificial insemination of queen bees are probable with frozen thawed

sperm (Collins, 2000; Cobey et al., 2013; Pallard et al., 2017 ).

Semen extender solution composition has a pivotal effect when overcoming the deleterious effects of ultra-low

temperatures and sustaining the fertilization viability of cryopreserved semen (Gul et al., 2017; Pallard et al., 2017; Alcay

et al., 2019a,b). Cryoprotectants are known as one of the essential ingredient to extend the shell-life of semen and are

used for preventing the detrimental effects of freezing, intracellular ice crystallization, and the effects of freeze-thaw

cycles (Watson and Fuller, 2001; Domingo et al., 2019). These molecules are classified as internal or penetrating and

external or non-permeating cryoprotectants (Salamon and Maxwell, 2000). Internal cryoprotectants increase the plasma

membrane fluidity, enhance the dehydration of spermatozoon, and reduce the intracellular ice formation, thereby

improving the freezing ability and survivability of spermatozoa (Holt, 2000 ).

On the other hand, external cryoprotectants act as additional solute and decrease the freezing temperature of

extender solution, reduce extracellular ice formation, and protect spermatozoon against cryopreservation as well (Amann,

1999). Out of all of these cryopotectants, dimethyl sulfoxide (DMSO) has attracted the most attention because to date it

provides the best post-thaw motility and viability results for honey bee spermatozoa cryopreservation (Taylor et al., 2009;

Hopkins ad Herr, 2010; Wegener et al., 2014; Alcay et al., 2016, Alcay at al., 2019a, b); Yaniz et al., 2019). However,

there are other cryoprotective agents (CPAs), which are commonly used to protect survival of spermatozoa at low

191

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.

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

serum albumin, gelatin, peptones and sericin) have been recently considered instead (Hubalek, 2003; Igbal et al., 2016;

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

Animals

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.

Chemicals

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

Semen was collected by squeezing drone abdomens as described by Collins and Donoghue (1999) using a Schley

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 3L of semen (approximately

3-4 mature drones) for each freezing group.

Sperm motility

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

motility respectively (Nur et al., 2012 ).

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, NaHCO₃0.21g, KCl 0.04g, Amoxicillin

0.03g, Catalase 200L 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 (3L) 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 -196^C. 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).

Statistical analyses

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.

RESULTS

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

192

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.

groups. No differences were observed across the diluted semen plasma membrane functional integrity of used groups

(Figure 1).

Although freeze-thaw cycle affect the post-thaw sperm motility and plasma membrane integrity for used groups,

spermatozoa had greater post-thaw motility and plasma membrane integrity when frozen in trehalose supplemented

extender in comparison when frozen in Trehalose free extender. The freeze-thawing process was less harmful on drone

semen plasma functional membrane integrity, compared to sperm motility especially in trehalose-supplemented group.

Post-thaw sperm motility of Trehalose supplemented groups were better than the Trehalose free group (P>0.05). Also

post-thaw plasma membrane integrity of the 0.1M Trehalose supplemented group (91%) was better than the 0.05

Trehalose (89%) (P>0.05), and Trehalose free group (79%) (P<0.05) (Figure 2).

Figure 1 - Diluted semen motility (%), and plasma membrane functional integrity (%). ^a,b:Groups with different superscripts

for same parameters are significantly different (P<0.05).

Figure 2 - Post-thaw semen motility (%), and plasma membrane functional integrity (%). ^a,b: Groups with different

superscripts for same parameters are significantly different (P<0.05).

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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.

DISCUSSION

Semen preservation requires a decrease or interruption of spermatozoa metabolism which is realized with

cryopreservation. This technique includes diluting, cooling, freezing, and storing the sperm at -196°C in liquid nitrogen,

followed by the thawing of the stored sperm. However, the processes of cryopreservation can affect sperm motility,

viability and fertility; permanently or temporarily. Irreversible reduction of spermatozoa metabolism originates from the

dilution effect, cold shock treatment, the formation of intracellular ice crystals, osmotic stress, and lipid peroxidation that

can occur throughout the cryopreservation process (Isachenko, 2003; Domingo et al., 2019 ).

The extenders used includes, carbohydrates (trehalose), salts (Na Citrate, NaHCO3, KCl), enzyme (catalase),

cryoprotectant (DMSO, trehalose) and antibiotics. These chemicals interact with different part of spermatozoa and lead to

osmotic and toxic stresses, and induce biochemical changes in cell metabolism immediately after dilution (Ak et al.,

2010; Domingo et al., 2019; Yániz et al., 2019). There were no differences among diluted sperm motility and plasma

membrane functional integrity within studied groups generally. The extender ingredients, dilution time, rate,

temperatures, cooling to 5°C and freezing curve and presence of cryoprotectant also affect post-thaw semen quality (Ak

at al., 2010; Nur at al., 2012; Alcay et al., 2015; Alcay et al., 2019a,b ). The cryoprotectants that were used for preventing

the detrimental effects of cooling, intracellular ice crystallization throughout the freezing and thawing processes are

known as one of the essential ingredients of semen extenders (Watson and Fuller, 2001; Domingo et al., 2019). Post-

thaw sperm motility (53%) and plasma membrane integrity (79%) of control group were lower than diluted semen motility

(70%; P<0.01) and plasma membrane integrity (90%; P<0.01). The transitional population of immotile sperm with intact

plasma membranes reveals that these sperms lost mobility characteristics, but still had functional membranes. These

findings indicate that using extender solutions have an ability to preserve post-thaw drone sperm plasma membrane

integrity to some degree. Therefore, it appears that post-thaw drone plasma membrane integrity can withstand stress

more than the motility ability of the sperm when subjected to freezing related damages (Alcay et al., 2015). Casper et al.

(1996) indicated that less than 50% of sperms have intact membranes in samples with complete astenozoospermia.

Cryoprotective agents including the widely used compound DMSO that was used in the freezing media have

significant cytotoxic effects on spermatozoa, both in short term and long term storage. They can induce some factors from

mitochondria to nucleus and poly-(ADPribose)-polymerase (PARP) activation that cause translocation of apoptosis

(Hanslick et al., 2009 ). In addition, DMSO affects cell plasma membrane structures and induces pore formation (Notman

et al., 2006). As a cryoprotective agent DMSO yields better post-thaw sperm quality in general and for this reason it has

become one of the main extender ingredients in many studies to date (Paillard et al., 2017; Gül et al., 2017; Alcay et al.,

2019a,b ).

The present experiments were designed to improve post-thaw drone semen quality by adding different

concentrations of Trehalose (0.05M, 0.1M or none at all) in freezing extender with 12% DMSO. The addition of 0.05M

Trehalose to the extender with 12% DMSO leads to a small increase in recovery of post-thaw motility (P>0.05) and plasma

membrane integrity (P<0.05), but when added at a 0.1M concentration this led to even better post-thaw motility (P>0.05)

and plasma membrane integrity (P<0.05). Trehalose has specific interactions with sperm membrane phospholipids and

minimizes the degree of cell damage during the freeze-thaw cycle by increasing the extender solution osmolarity (Storey

et al., 1998; Solocinski et al., 2017). For example, supplementation of 0.2-0.6M Trehalose to media containing 10%

DMSO improves post-thaw cell viability in several mammalian cell types (Beattie et al., 1997, Buchanan et al., 2004 ).

However, the exact mechanism(s) by which Trehalose cryoprotects the cellular structures at ultra-low temperatures

remains unknown. We observed that the addition of 0.05 and 0.1M Trehalose to the freezing extender solution containing

12% DMSO, increased post-thaw drone sperm plasma membrane integrity as well (Figure 2).

CONCLUSION

In conclusion, the freeze-thaw cycle is detrimental to post-thaw drone semen viability. The addition of 0.1 or 0.05M

Trehalose to freezing media containing 12% Dimethyl Sulfoxide has been demonstrated to improve both post-thaw cell

motility and plasma membrane integrity. More research is needed, to improve post-thaw sperm quality with other possible

candidate substances in the future for honey bee artificial insemination applications.

DECLERATIONS

Corresponding author:

Email: icakmak@uludag.edu.tr

Authors’ contributions

Prof. Dr. Z. Nur performed watering, freezing, thawing, evaluation, statistical analysis and writing manuscript, PhD

student S. Çakmak collected semen from drones, Prof. Dr. İ. Çakmak reared, collected drones, interpreted data,

contributed to manuscript writing and revisions, E. Gokçe performed watering, freezing, thawing and evaluation, Assoc.

Prof. Dr. B. Ustuner and S. Alçay performed thawing, evaluation, statistical analysis, and manuscript writing, Research

assistants N. Onder and M. Toker performed freezing of semen, Prof. Dr. M Soylu contributed to manuscript writing.

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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.

Acknowledgments

This work was supported by TUBİTAK, Project Number: TOVAG215O586.

Competing interests

Authors declare no conflict of interest.

REFERENCES

Ak K, Cirit U, Nur Z, Bacınoğlu S, Pabuccuoğlu S, Özdaş ÖB and Birler S (2010). Effects of extender osmolarity, cooling rate, dilution rate and

glycerol addition time on post-thaw ram semen characteristics and fertilization. Istanbul Universitesi Veteriner Fakultesi Dergisi, 36 (2): 33-

46. https://actavet.org/Content/files/sayilar/26/33-46.pdf

Alcay S, Cakmak S, Cakmak I, Mulkpinar E, Gokce E, Ustuner B, Sen H and Nur Z (2019). Successful cryopreservation of honey bee drone

spermatozoa with royal jelly supplemented extenders. Cryobiology, 87: 28-31. DOI: https://doi.org/10.1016/j.cryobiol.2019.03.005

Alcay S, Cakmak S, Cakmak I, Mülkpinar E, Toker MB, Üstüner B, Sen H and Nur Z (2019). Drone Semen Cryopreservation with Protein

Supplemented TL-Hepes Based Extender. Kafkas Universitesi Veteriner Fakultesi Dergisi, 25(4): 553-557.

http://vetdergikafkas.org/uploads/pdf/pdf_KVFD_2485.pdf

Link:

Alcay S, Ustuner B and Nur Z (2016). Effects of low molecular weight cryoprotectants on the post-thaw ram sperm quality and fertilizing ability.

Small Rumınant Research, 136: 59-64. DOI: https://doi.org/10.1016/j.smallrumres.2016.01.009

Alçay S, Üstüner B, Cakmak İ, Cakmak S and Nur Z (2015). Effects of Various Cryoprotective Agents on Post-Thaw Drone Semen Quality. Kafkas

Universitesi Veteriner Fakultesi Dergisi, 21 (1): 31-35. Link: http://vetdergikafkas.org/uploads/pdf/pdf_KVFD_1702.pdf

Amann RP. (1999). Cryopreservation of sperm. In: Knobil E and Neill JD (Eds). Encyclopedia of reproduction. Academic Press, Burlington, MA, pp

773–783.

Beattie GM, Crowe JH, Lopez AD, Cirulli V, Ricordi C and Hayek A (1997). Trehalose: a cryoprotectant that enhances recovery and preserves

function of human pancreatic islets after long-term storage. Diabetes, 46 (3): 519-523. DOI: https://doi.org/10.2337/diab.46.3.519

Buchanan SS, Gross SA, Acker JP, Toner M, Carpenter JF and Pyatt DW (2004). Cryopreservation of stem cells using trehalose: evaluation of the

method

using

human

hematopoietic

cell

line.

Stem

Cells

Development,

(3):

295-305.

DOI:

https://doi.org/10.1089/154732804323099226 .

Çakmak I and Çakmak SS (2016). Beekeeping and Recent Colony Losses in Turkey. Uludag Aricilik Dergisi, 16 (1): 31-48. Link:

https://dergipark.org.tr/tr/download/article-file/405030

Casper RF, Meriano JS, Jarvi KA, Cowan L and Lucato ML (1996). The hypo-osmotic swelling test for selection of viable sperm for

intracytoplasmic sperm injection in men with complete asthenozoospermia. Fertility and sterility, 65 (5): 972-976. DOI:

https://doi.org/10.1016/S0015-0282(16)58271-5

Cobey SW, Tarpy DR and Woyke J (2013). Standard methods for instrumental insemination of Apis mellifera queens. Journal of Apicultural

Research, 52 (4):1-18. DOI: https://doi.org/10.3896/IBRA.1.52.4.09 .

Collins AM (2000). Relationship between semen quality and performance of instrumentally inseminated honey bee queens. Apidologie, 31 (3):

421-429. DOI: https://doi.org/10.1051/apido:2000132

Domingo P, Olaciregui M, González N, De Blas I and Gil L (2019). Comparison of different semen extenders and cryoprotectant agents to

enhance cryopreservation of rabbit spermatozoa. Czech Journal of Animal Science, 64(2): 59-66. DOI: https://doi.org/10.17221/53/2018-

CJAS

Gül A, Şahinler N, Onal AG, Hopkins BK and Sheppard WS (2017). Effects of diluents and plasma on honey bee (Apis mellifera L.) drone frozen-

thawed semen fertility. Theriogenology, 101:109-113. DOI: https://doi.org/10.1016/j.theriogenology.2017.06.020

Hanslick JL, Lau K, Noguchi KK, Olney JW, Zorumski CF, Mennerick S and Farber NB (2009). Dimethyl sulfoxide (DMSO) produces widespread

apoptosis

the

developing

central

nervous

system.

Neurobiology

Disease,

(1):

1-10.

DOI:

https://dx.doi.org/10.1016%2Fj.nbd.2008.11.006

Holt WV (2000). Basic aspects of frozen storage semen. Animal Reprodion Science, 62 (1-3): 3-22. DOI: https://doi.org/10.1016/s0378-

4320(00)00152-4

Hopkins BK and Herr C (2010). Factors affecting the successful cryopreservation of honey bee spermatozoa. Apidologie, 41 (5): 548-556. DOI:

https://doi.org/10.1051/apido/20010006

Hubalek

(2003). Protectants used in the cryopreservation of microorganisms. Cryobiology, 46 (3): 205-229. DOI:

https://doi.org/10.1016/s0011-2240(03)00046-4

Iqbal S, Andrabi SMH, Riaz A, Durrani AZ and Ahmad N (2016).Trehalose improves semen antioxidant enzymes activity, post-thaw quality, and

fertility

Nili

Ravi

buffaloes

(Bubalusbubalis).

Theriogenology,

85(5):

954-959.

DOI:

https://doi.org/10.1016/j.theriogenology.2015.11.004

Isachenko E (2003). Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present

success. Reproductive Biomedicine Online, 6 (2): 191-200. DOI: https://doi.org/10.1016/s1472-6483(10)61710-5

Moritz FA and Erler S (2016). Lost colonies found in a data mine: Global honey trade but not pests or pesticides as a major cause of regional

honeybee colony declines. Agriculture, Ecosystems & Environment . 216 , (15): 44-50, doi: https://doi.org/10.1016/j.agee.2015.09.027

Mosca F, Madeddu M, Sayed AA, Zaniboni L, Iaffaldano N and Cerolini S (2016). Data on the positive synergic action of dimethylacetamide and

trehalose on quality of cryopreserved chicken sperm. Data in Brief, 9: 1118-1121. DOI: https://doi.org/10.1016/j.dib.2016.11.059

Neumann

and Carreck NL (2010).Honey bee colony losses. Journal of Apicultural Research, 49 (1), 1-6. DOI:

https://doi.org/10.3896/IBRA.1.49.1.01

Notman R, Noro M, O'Malley B and Anwar J (2006). Molecular basis for dimethylsulfoxide (DMSO) action on lipid membranes. Journal of

American Chemical Society, 128 (43): 13982-13983. DOI: https://doi.org/10.1021/ja063363t

Nur Z, Cakmak S, Ustuner B, Cakmak I, Erturk M, Abramson CI, Sağırkaya H and Soylu MK (2012). The use of hypo-osmotic swelling test, water

test, and supravital staining in the evaluation of drone sperm. Apidologie, 43 (1): 31-38. DOI: https://doi.org/10.1007/s13592-011-0073-

Paillard M, Rousseau A, Giovenazzo P and Bailey JL (2017). Preservation of domesticated Honey Bee (Hymenoptera: Apidae) drone semen.

Journal of Economic Entomology, 110(4), 1412-1418. DOI: https://doi.org/10.1093/jee/tox149

195

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.

Salamon

and Maxwell WM (2000). Storage of ram semen. Animal Reproduction Science, 62 (1-3): 77-111. DOI:

https://doi.org/10.1016/s0378-4320(00)00155-x

Solocinski J, Osgood Q, Wang M, Connolly A, Menze MA and Chakraborty N (2017). Effect of trehalose as an additive to dimethyl sulfoxide

solutions

ice

formation,

cellular

viability,

and

metabolism.

Cryobiology,75:

134-143.

DOI:

https://doi.org/10.1016/j.cryobiol.2017.01.001

Storey BT, Noiles EE and Thompson KA (1998). Comparation of glycerol, other polyols, trehalose, and raffinose to provide a defined

cryoprotectant medium for mouse sperm cryopreservation. Cryobiology, 37 (1): 46-58. DOI: https://doi.org/10.1006/cryo.1998.2097

Taylor M, Guzmán A, Novoa E, Morfin N and Buhr MM (2009). Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with

selected

diluents,

cryoprotectants,

and

semen

dilution

ratios.

Theriogenology,

(2):

149-159.

DOI:

https://doi.org/10.1016/j.theriogenology.2009.02.012

Watson PF and Fuller BJ (2001) Principles of Cryopreservation of Gametes and Embryos, in: Watson, P. F., Holt, W. V. (Eds.), Cryobanking The

Genetic Resource: Wildlife Conservation for the Future? CRC Press, London, pp. 21-46.

Wegener J, May T, Kamp G and Bienefeld K (2014). A successful new approach to honeybee semen cryopreservation. Cryobiology, 69 (2): 236-

242. DOI: https://doi.org/10.1016/j.cryobiol.2014.07.011

Yániz J, Palacín I, Santolaria P (2019). Effect of chamber characteristics, incubation, and diluent on motility of honey bee (Apis mellifera) drone

sperm. Apidologie, 50(4): 472-481. DOI: https://doi.org/10.1007/s13592-019-00659-y

196

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.