Elsevier

Animal Reproduction Science

Volume 162, November 2015, Pages 37-42
Animal Reproduction Science

Effectiveness of glucose–methanol extender for cryopreservation of Huso huso spermatozoa

https://doi.org/10.1016/j.anireprosci.2015.09.005Get rights and content

Highlights

  • Glucose concentration is important for the cryopreservation of beluga sturgeon sperm.

  • Sperm of beluga sturgeon was effectively cryopreserved with glucose–methanol.

  • The optimal cryopreservation conditions were found to be 0.2 M glucose in the extender.

Abstract

The present approach was designed to evaluate the methanol–glucose extender effects on sperm cryopreservation in beluga sturgeon, Huso huso. Sperm quality was examined by measuring post-thaw sperm motility and fertilizing rate at hatching stage. We first tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30 M) in a methanol extender on post-thaw sperm motility. The optimal cryopreservation conditions were found to be 0.2 M glucose in the extender. Then, motility and fertilization rates of sperm cryopreserved with 0.2 M glucose and 10% methanol (GM) were compared to Tris–sucrose–KCl in 10% methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 15 and 30 min equilibration in GM extender before and after cryopreservation were measured. Higher post-thaw sperm motility duration and percentage as well as fertilization rate were obtained with the GM extender when compared to TSKM extender. Equilibration of sperm in extender did not affect the motility quality of either fresh-diluted or frozen/thawed sperm, while fertilization rate showed a significant decline alone after 30 min of post-thaw storage. Our results indicated that the use of a simple extender consisting of 0.2 M glucose in 10% methanol can be an alternative cryopreservation method to those previously described for sturgeons.

Introduction

Most endangered and commercially valuable fish species, such as sturgeons are in a great need of aquacultural restocking, which relies on artificial reproduction (Havelka et al., 2011). Cryopreservation is a promising way for preservation of rare and endangered species of sturgeon. It plays an important role in transporting genetic material between facilities, optimal use in aquaculture, reducing the risk of spreading infections, performing hybridization studies, protecting gene pool, conserving biodiversity, selective breeding activities, and protecting endangered species (Sarder et al., 2012, Kutluyer et al., 2014).

Early studies on sturgeon sperm cryopreservation were carried out in the 1960s (Burtsev and Serebryakova, 1969) with limited success. Since the 90s, methods resulting in sufficient sperm post-thaw motility have been described (Glogowski et al., 2002, Psenicka et al., 2008). The latter methods employed extenders consisting of potassium ions and sucrose buffered by Tris–HCl in methanol. Recently, simple extenders containing glucose and methanol have been used for cryopreservation of salmonid fish (Ciereszko et al., 2014, Dietrich et al., 2014, Nynca et al., 2014) and also Siberian sturgeon, Acipenser baerii (Judycka et al., 2015) sperm. Furthermore, some studies about the short and long-term storage of Huso huso sperm have been conducted (Aramli, 2014, Aramli et al., 2015). Despite these advances, many issues still need to be studied in order to identify efficient and simple protocols for cryopreservation of this species sperm.

Cryopreservation involves several parameters that need be stabilized to improve post-thaw survival. These factors include cryoprotectant type and concentration (Lahnsteiner et al., 1996), equilibration time (Babiak et al., 2001, Perez-Cerezales et al., 2010), and number of spermatozoa (Ciereszko et al., 2013). This study was conducted to test the effectiveness of the glucose–methanol extender for the cryopreservation of sperm of H. huso. In the first experiment we aimed to optimize glucose concentration in the extender. In the second experiment sperm motility parameters and fertilizing ability of sperm cryopreserved in 0.2 M glucose and 10% methanol (GM) were compared to Tris–sucrose–KCl–methanol extender (TSKM). Additionally, we examined sperm motility and fertilization rate in relation to 30 min equilibration in GM extender and 30 min of post-thaw storage.

Section snippets

Collection of gamete and measurements of sperm density and seminal plasma osmolality

The study was performed at the Sturgeon Hatchery Center in Mazandaran, Iran. Broodfish (body weight 40–45 kg) were selected from wild breeders that originated from the Caspian Sea. Before stimulation, the fish (both male and female) were transferred from ponds to tanks with a water temperature of 15–16 ˚C, an oxygen content of >5 mg/L and a pH of 7.6–7.9. Next, the males were stimulated hormonally with an injection of 5 μg kg−1using a synthetic analog LHRH-A2 (Ningbo Renjian Pharmaceutical Group

Effect of glucose concentration in extender containing 10% methanol on sperm motility parameters of fresh and frozen/thawed sperm

The results of comparing different concentrations of glucose on fresh and post-thaw beluga sperm parameters are presented in Fig. 1(A) and (B). Glucose concentration in the extender did not have any effect on the fresh-diluted sperm (∼ 85%) but significantly affected post-thaw sperm motility parameters. The highest post-thaw sperm duration (48 s) and percentage (50%) was observed at 0.2 M glucose concentration. Therefore, this concentration was selected to be used in subsequent experiments.

Effects of extender composition on sperm motility and fertilization rate of fresh and frozen/thawed sperm

The

Discussion

External cryoprotectants that do not permeate sperm cells often are used to protect sperm from damage during the freezing and thawing process. In fish sperm, the effects of egg yolk (Babiak et al., 2001), low density lipoproteins (Perez-Cerezales et al., 2010), soybean proteins (Cabrita et al., 2001), and bovine serum albumin (Stoss and Holtz, 1983) have been examined. Although these non-permeating cryoprotectants usually have a positive effect, high variability in the results often is

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.

We confirm that we

References (30)

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    The efficacy of glucose (monosaccharide) and sucrose (disaccharide) as cryoprotective supplements have shown particular promise, as these typically increase cell survival after freeze thawing. These sugars have also been widely applied in the cryopreservation of sturgeon semen (Tsvetkova et al., 1996, Billard et al., 2004, Boryshpolets et al., 2011; Judycka et al., 2015; Aramli et al., 2015). In the case of other disaccharides, trehalose is found in a number of plants and animals that can resist dehydration or freezing; it can form hydrogen bonds with the polar head groups of phospholipids and therefore helps to prevent fusion events in juxtaposed membranes (Woelders et al., 1997).

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