<abstract xmlns="http://www.w3.org/1999/xhtml">

<sec id="j_jvetres-2023-0001_s_005"><h3>Introduction</h3>
<p>Bees are currently artificially inseminated on a large scale for breeding and research purposes. The sperm of bees has a complex and varied structure, and determination of specific morphological defects in it is very difficult. Its comprehensive analysis by inspecting morphology and morphometry is an important tool for improving honey bee lines. The staining technique should interfere with the cells as little as possible while clearly showing the boundaries of the head and other elements. In this study, a comparative analysis of the morphometry of sperm was performed with various techniques for staining drone semen.</p>
</sec>
<sec id="j_jvetres-2023-0001_s_006"><h3>Material and Methods</h3>
<p>Semen was collected from 150 sexually mature Buckfast bee drones by artificially everting the copulatory organ. The morphology and morphometry of the sperm were assessed on slides prepared by three staining methods according to the protocols described online, using the Sperm Class Analyzer system. The lengths of the acrosome, nucleus, head in total, midpiece, tail without midpiece, tail with midpiece, and entire sperm were measured.</p></sec>
<sec id="j_jvetres-2023-0001_s_007"><h3>Results</h3>
<p>The most details of the drone sperm structure could be seen when stained with the eosin-nigrosin complex. This method made it possible to identify all structures and revealed the uneven distribution of sperm proteins in different parts of the tail. With the Sperm Stain method fewer details of the sperm structure were recognisable, and the fewest were with SpermBlue.</p></sec>
<sec id="j_jvetres-2023-0001_s_008"><h3>Conclusion</h3>
<p>The staining method, and thus the chemical reagents used, affect the dimensions of drone sperm. Given the great research potential of modified spermatozoa of insects, a standard for slide preparation for the evaluation of morphological and morphometric semen parameters should be established, as this would facilitate result comparison between laboratories and increase the value of morphological analysis of sperm for predicting and assessing fertility.</p>
</sec></abstract>

Introduction
Bees are currently artificially inseminated on a large scale for breeding and research purposes. The sperm of bees has a complex and varied structure, and determination of specific morphological defects in it is very difficult. Its comprehensive analysis by inspecting morphology and morphometry is an important tool for improving honey bee lines. The staining technique should interfere with the cells as little as possible while clearly showing the boundaries of the head and other elements. In this study, a comparative analysis of the morphometry of sperm was performed with various techniques for staining drone semen.

Material and Methods
Semen was collected from 150 sexually mature Buckfast bee drones by artificially everting the copulatory organ. The morphology and morphometry of the sperm were assessed on slides prepared by three staining methods according to the protocols described online, using the Sperm Class Analyzer system. The lengths of the acrosome, nucleus, head in total, midpiece, tail without midpiece, tail with midpiece, and entire sperm were measured.
Results
The most details of the drone sperm structure could be seen when stained with the eosin-nigrosin complex. This method made it possible to identify all structures and revealed the uneven distribution of sperm proteins in different parts of the tail. With the Sperm Stain method fewer details of the sperm structure were recognisable, and the fewest were with SpermBlue.
Conclusion
The staining method, and thus the chemical reagents used, affect the dimensions of drone sperm. Given the great research potential of modified spermatozoa of insects, a standard for slide preparation for the evaluation of morphological and morphometric semen parameters should be established, as this would facilitate result comparison between laboratories and increase the value of morphological analysis of sperm for predicting and assessing fertility.

Identification of honey bee sperm structures following the use of various staining techniques

Journal of Veterinary Research

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Introduction
Bees are currently artificially inseminated on a large scale for breeding and research purposes. The sperm of bees has a complex and varied...

	Staining technique

	Eosin- nigrosin	Sperm Stain	SpermBlue	Complex of eosin and gentian violet	Complex of eosin and gentian violet	Live/dead dual fluorescent staining method	Live/dead dual fluorescent staining method	Bengal rose stain	Bengal rose stain	Haematoxylin and eosin
Morphometric parameter
	Notes (reference)

				Season beginning, June (19)	Season end, August (19)	Large and small drone serum sampled from queenright (14)	Laying worker colonies (14)	Brazil, rainy season (36)	Brazil, dry season (36)	(42)
Acrosome length (μm)	3.74a ± 0.61	3.59b ± 0.45	-	4.73 ± 0.32	4.66 ± 0.32	-	-	-	-	3.58 ± 1.21

Nucleus length (μm)	4.25a ± 0.37	4.06b ± 0.23	-	4.78 ± 0.25	4.69 ± 0.25	-	-	-	-	4.44 ± 0.61

Head length (μm)	8.08a ± 0.40	7.85b ± 0.29	7.71b ± 0.53	9.43 ± 0.38	9.35 ± 0.39	-	-	9.48 ± 0.10	9.24 ± 0.10	7.85 ± 0.65

Midpiece length (μm)	30.63 ± 0.87	-	-	-	-	-	-	-	-	-

Flagellum length (μm)	204.31 ± 10.12	226.16* ± 9.53	221.56* ± 11.29	264.07* ± 16.57	248.62* ± 16.38	-	-	253.47 ± 1.20	242.99 ± 4.00	222.96 ± 17.15

Total length (μm)	242.59a ± 11.54	232.33a ± 11.01	226.56a ± 12.03	273.50 ± 16.58	257.97 ± 16.37	242.0 ± 0.65	241.4 ± 0.34	262.92 ± 1.20	252.24 ± 4.10	230.81 ± 17.22

Identification of honey bee sperm structures following the use of various staining techniques

Published Online: Feb 04, 2023

Page range: 131 - 138

Received: Oct 08, 2022

Accepted: Jan 19, 2023

DOI: https://doi.org/10.2478/jvetres-2023-0001

Keywords
drone, sperm morphometry

© 2023 D. Banaszewska and K. Andraszek, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Morphometric variables of the sperm measured by the Sperm Class Analyzer and manually

Identification of honey bee sperm structures following the use of various staining techniques

Published Online: Feb 04, 2023

Page range: 131 - 138

Received: Oct 08, 2022

Accepted: Jan 19, 2023

DOI: https://doi.org/10.2478/jvetres-2023-0001

Keywordsdrone, sperm morphometry

© 2023 D. Banaszewska and K. Andraszek, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Morphometric variables of the sperm measured by the Sperm Class Analyzer and manually

Keywords
drone, sperm morphometry