REPORT NO. 2772
SUPRA-ZERO STORAGE OF HONEY BEE SPERM –
UPDATE FOR RAINBOW HONEY AND NELSON
HONEY LTD, SEPTEMBER 2015
CAWTHRON INSTITUTE | REPORT NO. 2772
SEPTEMBER 2015
SUPRA-ZERO STORAGE OF HONEY BEE SPERM –
UPDATE FOR RAINBOW HONEY AND NELSON
HONEY LTD, SEPTEMBER 2015
SEREAN ADAMS
Prepared for Rainbow Honey and Nelson Honey Ltd
CAWTHRON INSTITUTE
98 Halifax Street East, Nelson 7010 | Private Bag 2, Nelson 7042 | New Zealand
Ph. +64 3 548 2319 | Fax. +64 3 546 9464
www.cawthron.org.nz
APPROVED FOR RELEASE BY:
Type name
Jacquie Reed
Signature required
ISSUE DATE: 25 September 2015
RECOMMENDED CITATION: Adams SL 2015. Supra-zero storage of honey bee sperm – update for Rainbow Honey and
Nelson Honey Ltd, September 2015. Cawthron Report No. 2772. 13 p. plus appendices.
© COPYRIGHT: Cawthron Institute. This publication may be reproduced in whole or in part without further permission of the
Cawthron Institute, provided that the author and Cawthron Institute are properly acknowledged.
CAWTHRON INSTITUTE | REPORT NO 2772
SEPTEMBER 2015
INTRODUCTION
Cryopreservation (ultra-low temperature storage) and supra-zero storage methods (i.e.
storage at temperatures above 0°C) for sperm can be powerful tools in selective breeding.
The methods enable breeders to make the most desirable crosses on demand without
seasonal constraints and to evaluate the resulting progeny for a range of economically
important traits. With these methods in place, valuable germplasm can be stored and
recovered at will to optimally manage genetic diversity for long-term sustainability, to balance
selection intensity across traits and to manage genetic trade-offs between them.
In order for cryopreservation or supra-zero short-term storage to be implemented in selective
breeding, the methods developed must be reliable and robust. Ideally, queens inseminated
with stored sperm should produce functional hives and lay for at least several months.
However, the benchmark for implementing cryopreservation in selective breeding is the
ability to recover genes.
Several papers have been published relating to the development of a method for
cryopreserving honey bee sperm (e.g. Harbo 1979; Harbo 1981; Harbo 1983; Kaftanoglu and
Peng 1984; Stucky et al. 2008; Taylor et al. 2009; Hopkins and Herr 2010; Hopkins et al.
2012; Wegener and Bienfeld 2012; Wegener et al. 2014). However, with many of the
protocols evaluated, queens inseminated with cryopreserved sperm laid proportionately
variable numbers of workers (0-100%) and were often “patchy layers”. The number of sperm
reaching the spermatheca was also variable, sometimes by orders of magnitude, when
measured (e.g. Harbo 1979; Kaftanoglu and Peng 1984; Wegner et al. 2014) and where
reported, queens inseminated with cryopreserved sperm were absent after about two months
of laying (Hopkins et al. 2012).
Most cryopreservation methods dilute sperm with buffers containing cryoprotective agents
prior to freezing (chemicals that are almost always necessary for cells to survive freezing).
One of the problems with this is that dilution can cause activation of honey bee sperm which
may reduce longevity (Lensky and Schindler 1967); although there are some buffers with
high ionic concentrations that reversibly inhibit motility (Verma 1973; 1978). Dilution may also
cause breakdown of the extracellular matrix that ejaculated sperm are embedded in
triggering activation. The recent paper by Wegener et al (2014) overcame this by using
dialysis buttons to incorporate dimethyl sulphoxide as a cryoprotective agent into honey bee
sperm. This work is encouraging and further refinements to this research may help to
develop a method that is reliable and robust for honey bee sperm.
Supra-zero storage of sperm is a flexible alternative that provides a useful adjunct to
cryopreservation and is easier to carry out and maintain because no liquid nitrogen,
controlled freezing device or storage dewars are required. The bench mark for
implementation of a supra-zero storage method in selective breeding and for general use in
queen breeding is higher than for cryopreserved sperm. The method must enable the
recovery of functional hives and queens must be able to lay for at least several months.
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Although previous studies suggest that storage of undiluted semen is possible for several
weeks (Harbo 1974; Cobey et al. 2013), there is limited evidence in the literature
demonstrating how reliable this is in maintaining functional hives and the ongoing laying of
instrumentally inseminated queens with stored sperm.
Taber et al. (1979) dusted capillary tubes with streptomycin sulfate prior to semen collection
and then stored sperm under nitrogen gas at 13°C and had promising results after 113 days
storage. Hopkins (2014) further investigated the concept of pre-coating capillary tubes using
antimycotics in addition to antibiotics. Capillary tubes were loaded with a gelatin solution
containing the antibiotics and antimycotics and then placed in a Speed Vac until the contents
were desiccated, uniformly coating the inside of the capillary tube. Queens inseminated with
sperm stored for 439 days by this method at 13°C produced fertilized offspring (approx. 3095% worker brood, n=3). Queens inseminated with sperm stored for 45 days were less
variable in the proportion of fertilized off spring (approx. 70-100% worker brood, n=6). Other
studies have also investigated sperm storage at supra-zero temperatures (Poole and Taber
1970; Locke and Peng 1993; Collins 2000).
The aim of this study was to evaluate modifications to the method developed by Hopkins
(2014) to see if results could be further improved. Originally, we planned to evaluate a
number of treatments after varying periods of storage. However, a statistical power analysis
of Hopkins (2014) data suggested that a minimum of 32 queens was required per treatment
in order to be able to detect true differences between treatments. Therefore, we decided to
look at only four treatments, including the best treatment from Hopkins (2014) at one time
period only. We choose 14 weeks storage because this was greater than the time at which
queens in Hopkins study varied in terms of their ability to produce a high proportion of solid
worker brood. Increasing the concentration of antibiotics and incorporating antioxidants in
the coating mixture were evaluated as these were considered most likely to increase the
success of the method following personal communications with Brandon Hopkins
(Washington State University) and Jakob Wegner (Institute for Bee Research Hohen
Neuendorf, Germany) and from the literature antioxidants were reported as components of
spermathecal gland secretions (Weirich et al. 2002; Klenk et al 2004). We also choose to
perform two instrumental inseminations per queen rather than one because previous
research suggested that this increased the number of sperm reaching the spermatheca (for a
given volume of sperm) (Cobey 2007).
1. METHODS
1.1. Solutions and Micro-capillary coating
All chemicals were cell culture grade or higher and were sourced from Sigma (St Louis, MO).
Four treatment coating solutions were made up (Table 1). In contrast to Hopkins (2014),
coating solutions did not contain gelatin. This was because in preliminary work, solutions
containing 0.25 mg/100 mL, as used by Hopkins (2014) blocked. Reducing the gelatin
concentration to 0.125 mg/100 mL and 0.0625 mg/100 mL also resulted in blocking. Hopkins
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(2014) used larger 100 µL capillary tubes. However, this causes problems for statistics
because samples are not truly independent of one another.
Solutions were degassed for 30 min under vacuum before being aspirated into 5 µL capillary
tubes (Drummond Microcaps). Microcapillary tubes were then dried under vacuum. The
microcapillary tubes were visually inspected under light microscope and appeared to be
evenly coated. They were then stored under vacuum with desiccant at 4°C until ready to be
used.
Table 1. Treatment coating solutions. Antibiotics and antibiotics were dissolved and made up
to a final volume of 100 mL.
Treatment 1
(RED)
Ingredient
Penicillin (g)
Streptomycin (g)
Kanomycin (g)
Tylosin* (g)
Nystatin** (g)
Catalase***
Glutathione (g)
From
Hopkins
(2014)
0.05
0.044
0.06
0.0032
0.012
Treatment 2
(BLUE)
Treatment 3
(GREEN)
Increased
penicillin and
Antioxidants
streptomycin
added
0.25
0.05
0.22
0.044
0.06
0.06
0.0032
0.0032
0.012
0.012
10000 units
0.15366
Treatment 4
(BLACK)
Increased
penicillin and
streptomycin,
antioxidants
added
0.25
0.22
0.06
0.0032
0.012
10000 units
0.15366
*Tylosin made up at 100x concentration in Milli-Q water then 1 mL added to final coating solution to give final concentration of
0.0032 g/100 mL.
**Nystatin did not dissolve. Therefore maintained in suspension during loading into capillary tubes.
**Catalase concentration was 2000- 5000 units/mg. Therefore dissolved 29 mg in 100 mL and added 10 mL of this to final stock
to give 2.9 mg/100 mL or ~10000 units.
In addition to the coating solution, HHSBE solution was made up as reported by Cobey et al.
(2013) except that the catalase concentration was reduced to 0.0005 g as reported by
Hopkins (2014; Table 2).
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Table 2. Modified HHSBE solution. Part C ingredients were dissolved in 10 mL of Milli-Q
water then 1 mL of this solution was added to a 100 mL volumetric flask. Part A ingredients
were then added to the volumetric flasks and dissolved by adding Milli-Q water to a final
volume of 100 mL. Part B ingredients were then dissolved in 10 mL of the Part A+C solution
and the 0.9 mL of the Part B solution was added to the remaining 90 mL of Part A+C
solution.
Part A
Ingredient
Penicillin
Streptomycin
Kanamycin
TES
Tris Base
Sodium Phosphate Dibasic
Sodium Citrate
Arginine
Proline
Potassium Chloride
Sodium Chloride
NaHCO3
BSA (lipid rich)
Part B
Ingredient
Tylosin
EDTA
Glycine
Part C
Catalase
Amount (g)
0.05
0.044
0.06
0.6879
0.3633
0.0142
0.02942
0.01
0.05
0.61131
0.4847
0.042
0.002
0.032
0.003
0.0079
0.005
1.2. Semen Collection
Semen was collected from mature drones on 9-12 December 2014 using a Harbo syringe
that had been rinsed and filled with either RH diluent (supplied by Grant MacDonald) or
modified HHSBE diluent.
Sperm was collected by partially and then fully everting the endophallus and collecting the
exposed semen. Not all drones produced semen. If the semen came into contact with the
drone’s abdomen or the person collecting the semen, it was discarded because of
contamination.
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Semen was collected into a glass capillary tip and then drawn into the 5 µL coated
microcapillary tubes. The tip was washed between collections with either RH diluent or
HHSBE diluent and changed between microcapillary tubes. Each capillary tube was sealed
with petroleum jelly at each end and then placed into 4.5 mL cryovials that were gassed with
argon before being sealed and placed into an incubator set at 14°C within two hours of
collection. Different coating treatments were selected randomly for filling. As a control
treatment, semen was also collected into 5 µL microcapillary tubes that were not coated.
1.3. Instrumental Insemination
At the end of each collection day, sperm collected in microcapillary tubes that were not
coated was inseminated into virgin queens as controls. Five queens were inseminated for
each day of sperm collection. Queens were gassed with CO2 and then inseminated with the
collected semen using the Harbo syringe device and micromanipulators. The Harbo syringe
was used to push out the petroleum jelly plugs sealing the sperm inside the microcapillary.
Approximately 4.4 µL of sperm was inseminated into each queen and queens were
transferred into nucs overnight. In general, after 24 hours, queens were re-inseminated with
a second dose of sperm, although some queens were inseminated only once when
insufficient control sperm had been collected.
After 80-86 days of storage at 14°C, sperm stored in coated microcapillary tubes was
inseminated into virgin queens. In general, queens were gassed with CO2 and inseminated
with one micro-capillary of stored sperm then inseminated a second time approximately 24
hours later with a second micro-capillary tube of the same coating treatment. Microcapillary
tubes were wiped with 70% ethanol prior to being used for insemination. Inseminated queens
were placed into nucs and their ability to lay was determined by photographing the first frame
laid.
1.4. Brood Evaluation
For each queen, a photograph was taken of the capped brood on at least one side of a frame
approximately 7 weeks after the queens were inseminated (Appendix 1). Brood solidness
was scored subjectively by evaluating the percentage of sealed worker brood in a given area
of frame where 1 = <25% sealed worker brood present; 2 = 25-50% sealed worker brood
present; 3 = 50-75% sealed worker brood present and 4 = 75-100% sealed worker brood
present (Delaplane et al. 2013).
2. RESULTS
The number of queens surviving instrumental insemination was low, even for the controls
where sperm had not been stored for a maximum of 24 hours prior to insemination (Table 3).
Of the 20 queens that were inseminated as controls, 3 were inseminated once and 17 were
inseminated twice ~24 hours. Only five control queens survived by 21st January 2015 (25%).
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Of these, four were queens that had been inseminated twice and one queen had been
inseminated only once. The brood solidness score for control queens averaged 3.2.
A total of 78 queens were inseminated with stored sperm (Table 3). One queen was
inseminated with a microcapillary coated with Treatment 4 and then a microcapillary coated
with Treatment 3. However, this queen did not survive until laying. Survival of queens until
laying was similar to the control treatment ranging from 25-35% across treatments. Of the
queens that laid, those that were instrumentally inseminated with sperm stored in
microcapillaries coated with Treatment 4 (Black) had the highest brood solidness score
averaging 3.4. This was the treatment containing a higher concentration of antibiotics as well
as antioxidants and the brood solidness score was similar to that which was observed for
control queens.
Table 3. Summary of results showing number of queens inseminated in each treatment,
number of queens surviving until laying, percentage survival, brood solidness score range
and average brood solidness score*.
Number
of
Percentage
Number of
Queens
(%) Survival Brood
Brood
Queens
Surviving Following
Solidness Solidness
Instrumentally Until
Instrumental Score
score
Treatment
Inseminated
Laying
Insemination Range
average
Control
(Fresh Sperm)
20
5
25 2-4
3.2
Treatment 1
(Red)
20
6
30 1-4
2.2
Treatment 2
(Blue)
20
7
35 1-2
1.7
Treatment 3
(Green)
17
6
35 1-3
1.7
Treatment 4
(Black)
20
5
25 2-4
3.4
* 1 queen was also inseminated with one microcapillary of treatment 4 and the one of treatment 3. However this queen did not survive.
3. SUMMARY AND FUTURE DIRECTIONS
In this study, four coating treatments containing antibiotics and antifungal agents with and
without antioxidants were applied to microcapillary tubes to see if bee sperm could be
reliably stored for a period of ~12 weeks. Unfortunately, the percentage of queens surviving
the instrumental insemination process was low, even in the control treatments. We choose to
perform two inseminations per queen rather than one because previous studies had
demonstrated that the number of sperm reaching the spermathecae was higher when
queens were inseminated twice rather than once for a given total volume of sperm (Cobey
2007). An unintended consequence of this decision may have been an increased mortality
rate from the procedure. We are not aware of this information in the literature. We cannot
conclusively say that inseminating queens twice rather than once increased mortality
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because we do not have appropriate controls, although 2 of the 3 control queens
inseminated only once died before laying, implying that double instrumental inseminations
may not be solely responsible for the increased mortality rate observed here over what is
generally observed (50-80% survival). Future studies should focus on improving the survival
rate of instrumentally inseminated queens as well as determining whether there is a
difference in survival between queens that are inseminated twice and queens that are
inseminated only once.
The percentage of queens laying following instrumental insemination with stored sperm was
similar to what was observed for control queens. We did observe bacterial contamination in
some of the stored cryovials on the outside of the microcapillary tubes suggesting that some
oxygen remained in the cryovials or was able to leak in over time. We washed the outside of
the microcapillary tubes with 70% ethanol before inseminations so that this contamination
was not passed on to the sample. However, future studies should consider also wiping the
capillary tubes with 70% ethanol prior to storage and including O2 absorbing packs in the
storage container for the microcapillary tubes. Because the percentage of queens laying was
similar to the controls, we believe the effect of this contamination in this study was minimal.
The best coating treatment, in terms of brood solidness was Treatment 4 which contains 5x
higher penicillin and streptomycin than Hopkins (2014) used in his study as well as the
antioxidants, catalase and glutathione. Because the number of queens successfully
inseminated was low, future studies should consider repeating this treatment to determine its
reliability and robustness as a storage method. Studies should also consider varying the
levels of the antioxidants in the coating as well as adding additional antioxidants such as
superoxide dismutase and EDTA. It would also be interesting to know the effect of storage
on the production of reactive oxygen species by sperm. As well, the duration of laying should
also be considered and compared to inseminations performed with fresh sperm.
4. ACKNOWLEDGEMENTS
This work would not have been possible without Rae Butler and her dedicated team at
Rainbow Honey in particular, Britney who did a lot of the drone semen collection and the
control instrumental inseminations. I especially thank Rae for all her hard work and
dedication to this project. I also thank Brandon Hopkins for his insightful suggestions and
help in planning this work.
5. REFERENCES
Cobey SW, 2007. Comparison studies of instrumentally inseminated and naturally mated
honey bee queens and factors affecting their performance, Apidologie 38: 390-310.
Cobey SW, Tarpy DR, Woyke J, 2013. Standard methods for instrumental insemination of
Apis mellifera queens. Journal of Apical Research 52 (4): 1-18.
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Collins AM, 2000. Survival of honey bee (Hymenoptera: Apidae) spermatozoa stored at
above-freezing temperatures. Journal of Economic Entomology 93(3):568-71.
Delaplane KS, Van Der Steen J, Guzman E, 2013. Standard methods for estimating strength
parameters of Apis mellifera colonies. In V Dietemann; J D Ellis; P Neumann (Eds) The
COLOSS BEEBOOK, Volume I: standard methods for Apis mellifera research. Journal of
Apicultural Research 52(1): http://dx.doi.org/10.3896/IBRA.1.52.1.03.
Harbo JR, 1974. A technique for handling stored semen of honey bees. Annuals of the
Entomological Society of America 67: 191-194.
Harbo JR, 1979. Storage of honey bee spermatozoa at -196°C. Journal of Apicultural
Research 18(1): 57-63.
Harbo JR, 1981. Viability of honey bee eggs from progeny of frozen spermatozoa. Annals of
the entomological society of America 74(5):482-486.
Harbo JR, 1983. Survival of honey bee (Hymenoptera: Apidae) spermatozoa after two years
in liquid nitrogen (-196°C). Annals of the Entomological Society of America 76(5): 890-891.
Hopkins BK, 2014. Artificial reproductive techniques in honey bees: Sperm cell physiology,
semen collection and storage, PhD thesis, Washington State University, USA 109 pp.
Hopkins BK, Herr C, 2010. Factors affecting the successful cryopreservation of honey bee
(Apis mellifera) spermatozoa. Apidologie 41: 548-556.
Hopkins BK, Herr C, Sheppard WS, 2012. Sequential generations of honey bee (Apis mellifera)
queens produced using cryopreserved semen. Reprod Fertil Dev. 24(8): 1079-83.
Kaftanoglu O, Peng Y-S, 1984. Preservation of honey bee spermatozoa in liquid nitrogen.
Journal of Apical Research 23(3): 157-163.
Klenk M, Koeniger G, Koeniger N, Fasold H, 2004. Proteins in spermathecal gland secretion
and spermathecal fluid and the properties of a 29kDa protein in queens of Apis mellifera.
Apidologie 35: 371-381.
Lensky Y, Schindler H, 1967. Motility and reversible inactivation of honeybee spermatozoa in
vivo and in vitro. Ann. Abeille 10: 5-16.
Locke SJ and Peng YS, 1993. The effect of drone age, semen storage and contamination on
semen quality in the honey bee (Apis mellifera). Physiological Entomology 18 (2) 144-148.
Poole HK, Taber S, 1970. In Vitro Preservation of Honey Bee Semen Enhanced by Storage
at 13—15°C. Annuals of the Entomnological Society of America 63, 1673-1674.
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Stucky M, Hopkins BK, Herr C, 2008. Cryopreservation of honey bee spermatozoa.
Reproduction, Fertility and Development 20(1):127-128.
Taylor MA, Guzmán-Novoa E, Morfin N, Buhr MM, 2009. Improving viability of cryopreserved
honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen
dilution ratios. Theriogenology 72: 149-159.
Taber S, Poole HK, Edwards JF, 1979. Enhanced fertility of honey bee semen stored in vitro
and possibly a reversal of senescence. Apidologie 10(2): 129-136.
Verma LR, 1973. An ionic basis for a possible mechanism of sperm survival in the spermatheca of the
queen honey bee (Apis mellifera L.), Comparative Biochemistry and Physiology Part A Physiology
44(4): 1325-1331.
Verma LR, 1978. Biology of honeybee (Apis mellifera L.) spermatozoa.1. Effect of different diluents on
motility and survival. Apidologie 9(3):167-174.
Wegener J and Bienefeld K, 2012. Toxicity of cryoprotectants to honey bee semen and
queens. Theriogenology 77: 600-607.
Wegener J, May T, Kamp G, Bienefeld K, 2014. A successful new approach to honeybee
semen cryopreservation. Cryobiology 69: 236-242.
Weirich GF, Collins AM, Williams VP, 2002. Antioxidant enzymes in the honey bee, Apis
mellifera 33: 3 -14.
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6. APPENDIX
Appendix 1. Frame photos taken for each queen inseminated with either fresh sperm
or stored sperm.
Controls (fresh II)
Treatment 1
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Treatment 2
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Treatment 3
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Treatment 4
17