Freezability of buffalo semen with TRIS extender enriched with disaccharides (trehalose or sucrose) and different glycerol concentrations

Gamal A. El-Sisy, Mohamed I. Shahba, Reda I. El-Sheshtawy

1Animal Reproduction and Al Department, Veterinary Division, National Research Center, Dokki, Giza, Egypt

Freezability of buffalo semen with TRIS extender enriched with disaccharides (trehalose or sucrose) and different glycerol concentrations

Gamal A. El-Sisy1*, Mohamed I. Shahba1, Reda I. El-Sheshtawy1

1Animal Reproduction and Al Department, Veterinary Division, National Research Center, Dokki, Giza, Egypt

ARTICLE INFO

Article history:

Received 2016

Received in revised form 2016

Accepted 2016

Available online 2016

Buffalo

Trehalose

Sucrose

Glycerol

Extender

Objective:To display the effect of different concentrations of glycerol, as a cryopreservative, on the quality of the frozen-thawed buffalo semen extended in TRIS extender enriched with disaccharides (trehalose or sucrose).Methods:Semen samples were extended in Tris-Citric acid-Fructose-Egg yolk without addition of trehalose / sucrose and with 6.4 % glycerol as a control (TFEG-C) and with the addition of Trehalose / Sucrose and different concentrations of glycerol to ensure 60 million motile spermatozoa mL-1. Semen cooled slowly up to 5 and equilibrated for 4 h. Semen was packed into 0.25 mL polyvinyl French straws. The straws were placed horizontally on a rack and frozen in a vapor 4 cm above liquid nitrogen (LN2) for 10 min then dipped in liquid LN2. Frozen straws were thawed at 37 ℃ for 1 min. The parameters studied were sperm motility, sperm viability, sperm abnormality, sperm membrane integrity (HOST), percent of normal intact acrosome and DNA fragmentation.Results:The best sperm motility, sperm liveability, sperm abnormality, sperm cell membrane and DNA integrities appeared with TFES-G 5.5% (41.00% ± 2.08%, 70.40% ± 2.27%, 7.80% ± 1.19%, 68.10% ± 1.55%, 98.90% ± 0.50%, respectively ) and TFES-G 7.3% (41.50% ± 1.98%, 70.70% ± 2.03%, 10.80% ± 0.88%, 69.30% ± 1.85% and 96.40% ± 0.88%, respectively).Conclusion:From the present study, it can be concluded that addition of glycerol (5.5% or 7.3%) to Tris-Fructose-Egg yolk-Sucrose extender might help in improvement of the post-thawed characteristics of buffalo frozen semen.

1. Introduction

Artificial insemination with frozen semen is the leading and promising applied tools to improve buffalo breeding. Unfortunately, the unsatisfactory resulted conception rates of the buffalo frozen semen, is challenge, withhold its suitability in the field. During standard cryopreservation technique sperm cells are subjected to temperature alterations, cellular dehydration and freezing-thawing[1]. Moreover, cryopreservation processes is a major source for ROS generation that is accompanied with reduction in antioxidant systems resulting in compromised post-thawing semen quality and sperm and fertilizing ability[2-5].

Extenders composition and cooling, freezing and thawing programs are important factors affecting buffalo semen freezability and fertility[6]. Tris-based extenders are commonly used for semen preservation in most farm animals[7] including buffaloes. Cryoprotectants are either penetrating the sperm cells like glycerol or non-penetrating like sugars[8]. Although the high toxicity of glycerol, it is still the best penetrating cryoprotectant in extenders used for semen freezing. [9,10] and the optimization of glycerol levels in the extender can achieved by a balance between the toxic and protecting consequences [8].

In the latest years, trehalose was added to the extenders of bull[11,12], buffalo bulls[13,14], ram[15-18], goat[19], to improve post thawing semen quality. Trehalose may be serve as a cryoprotectant[20,21], as it supports cell dehydration, resultingin decreased water flow across the sperm membrane during cryopreservation[22] and prevent ice crystals production[23,24]. Trehalose interact with and modify sperm membrane phospholipids and proteins resulting in improved membrane flexibility that withstand cryo-injuries[15-23]. Inclusion of sucrose as nonpermeating cryoprotectant sugar in extenders seems to provide more protection to the sperm cells. Sucrose promotes cell dehydration before freezing, and prevents injury caused by intracellular ice formation[23-24].

Coupling of more than one cryoprotectants with different mode of action in semen freezing extender could be excellent than that of sole one[26]. Earlier studies postulated the synergistic protective action between trehalose and glycerol in bull and boar semen freezing extenders[27,28], although the postulated synergic effects between glycerol and trehalose in few experiments have assess this hypothesis[29-31].

The aim of the current study was to investigate the effect of various glycerol concentrations addition to fixed concentrations of trehalose and sucrose within a Tris-based extender on buffalo sperm parameters after the freeze-thawing process. Is trehalose or sucrose supplementation enable to reduce the glycerol levels in the extender and resulted in increased semen quality?

2. Materials and methods

Semen was obtained from 5 mature buffalo-bulls maintained at Abbasia semen Freezing Center, General Organization for Veterinary Services, Ministry of agriculture, Egypt, by using an artificial vagina (42 ℃). After initial evaluation, only semen samples with at least initial sperm motility of 70%, and normal sperm of 80% were used for further processing. Visual motility, sperm liveability, sperm abnormalities and sperm membrane integrity were assessed[32]. For extension, Tris-Fructose-Egg yolk-Glycerol (TFEG) was utilized as described by[33]. Semen samples were pooled to ovoid individual variations and divided into 7 aliquots; one extended with the basic extender (TFEG-C, control) and other aliquots were extended with TRIS-Fructose-Egg yolk-Trehalose / Sucrose (TFET/S) extender containing the different concentrations of glycerol according to Chaveiro,et al. [34] with some modifications, providing a final concentration of 60 ×106 sperm/mL, as shown in Table 1.

Table 1TRIS extender supplemented with different concentrations of glycerol (g/100 mL).

After freezing as described by El Sheshtawyet al.[12] and thawing in water bath (37 ℃) for 30 seconds., estimation of the post-thawed sperm motility, liveability, and abnormalities were done according to Campbell[35]. Sperm membrane integrity was evaluated by using the HOST[36] and DNA integrity by using Acridine orange staining technique[37] were adopted as described by Abdel Kader [38].

The obtained data were tabulated and computed for statistical analysis, where appropriate, according to the SAS? computerized program v. 9.2[39]. Mean ± SEM, ANOVA and LSD were calculated to deduce the effect and the most efficient concentrations of glycerol on the post-thawed characteristics of buffalo spermatozoa.

3. Results

The present results (Table 2) revealed significant differences in the post-thawed buffalo semen quality parameters after addition of various concentration of glycerol to sucrose enriched Tris-extender. The best sperm motility, sperm liveability, sperm abnormality, sperm cell membrane and DNA integrities appeared with TFES-G 5.5% (41.00% ± 2.08%, 70.40% ± 2.27%, 7.80% ± 1.19%, 68.10% ± 1.55%, 98.90% ± 0.50%, respectively ) and TFES-G 7.3% (41.50% ± 1.98%, 70.70% ± 2.03%, 10.80% ± 0.88%, 69.30% ± 1.85% and 96.40% ± 0.88%, respectively). Combination of glycerol and trehalose did not exert any significant affect on many of the studied post-thawing semen parameters except sperm membrane integrity that were significantly higher in all glycerol concentrations as compared with control. Based on the obtained results, it can be concluded that addition of glycerol (5.5% or 7.3%) to Tris-Fructose-Egg yolk-Sucrose enriched extender resulted in improvement of the post-thawed characteristics of buffalo frozen semen. Moreover, addition of different concentrations of glycerol to trehalose enriched extender provide better preservation for sperm membrane.

Table 2Effects of different glycerol concentrations on sperm assessment parameters of frozenzbuffalo semen (Means ± SEM).

4. Discussion

Reduction in motility, viability and fertility are the most common deleterious effects on sperm during cryopreservation owing to multifactorial cryo-damage[9]. Such cryodamage can be alleviatedby optimizing the types and levels of cryoprotectants added to the extender to protect the sperm cells during freezing[34,40,41]. Although several reports have evaluated the efficiency of trehalose cryoprotection at various concentrations in extenders, the possible synergistic cryoprotective action of trehalose and glycerol has not been fully studied up to date in buffalo semen[29,31,42]. This experiment was designed to estimate the optimum levels of glycerol and trehalose or sucrose in a Tris-based extender that exert synergistic cryoprtotection and yielded better post-thawing semen quality when used for buffalo bull semen cryopreservation. It is a trial to reduce the glycerol levels in the extender by trehalose or sucrose supplementation that could resulted better semen cryopreservation. In this study, no extra-beneficial action on all post thawing semen parameters could be noticed by adding different levels of glycerol to Trehalose enriched extender as compared with control. The experimental reports belonging to the effect of inclusion of trehalose in semen extender on post-thawing semen quality are paradoxal. Some studies indicated that trehalose addition to extender was not beneficial as post-thawing semen quality was not significantly improved in bull[43,44] stallion[45], deer[46], and gazelle [47]. Moreover, the addition of sucrose to bull semen extender exhibits deleterious effect on post-thawing sperm motility[48]. In contrast, trehalose improved the post-thawing semen quality in ram[15,23,24], goat [49,50] and rabbit [51]. Contradictory to our results, addition of 50 mM trehalose to bull freezing extender improved post-thawing semen quality[12]. Najafi,et al., [18] observed existence synergistic effect between both 5% glycerol and 100 mmol/L trehalose and 7% glycerol with 50 mmol/L trehalose in ram semen extender which offered excellent sperm protection that manifested by the better post-thawing sperm parameters than other combinations, or glycerol alone. Aisen,et al. [23] and Jafaroghli,et al.[25] noticed a better cryoprotective effects and a superior recovery of post-thawing ram spermatozoa with addition of 3% or 5%glycerol combined with 100 mmol/L trehalose in the extender. Khalili,et al. [41] observed that combining 200 mmol/L of trehalose (198.24 mmol/L) with 8% glycerol yielded the best protection and survival for goat semen during cryopreservation. These various reports clarify the differences between species regarding the optimum concentration of trehalose and glycerol in extender that offer the best protection of sperm cells during cryopreservation. Our findings revealed slight improvement in characteristics of buffalo frozen semen with sucrose and glycerol concentration of 5.5% and 7.3%, a finding which came in agreement with that observed in some earlier reports of Tambing,et al. [52]; Farshad,et al[53] and Qureshi,et al. [54] in buck semen; Corcuera,et al. [55] in boar semen; Miclea,et al. [56] in ram semen; Ta?demir,et al. [57] in bull semen. Glycerol is the most common and efficient cryprotective agent in extender and its concentration is crucial for the success of semen cyropresevtion in farm animals [58,59]. The improvement of semen preservability in our results might be due to the link of glycerol to intracellular water of the spermatozoa that prevent ice crystals production which have a deleterious and damaging action on sperm membrane during cryopreservation [8,60,61].

In conclusion, the current study revealed a synergistic action of addition different concentration of glycerol to sucrose enriched extender on buffalo bull spermatozoa survivability during cryopreservation. This synergism could not be observed between trehalose and glycerol. The top post-thawing quality was noticed with combination of 5.5 and 7.3% glycerol and 4.96% sucrose in freezing extender. It seems being suggested to test other combinations of sucrose, trehalose and glycerol and any of other cryoprotectants and exploring other concentration ranges to formulate a suitable extender for buffalo bull semen.

Conflict of interest statement

The authors declare that they have no conflict of interest.

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ment heading

10.1016/j.apjr.2016.07.007

*Corresponding author: Gamal A. El-Sisy, Animal Reproduction and AI department, Veterinary Research Division, National Research Centre, Dokki, Giza, Egypt .

Tel: 202-01121410020; 202 33371635

Fax : 202-3760187

E-Mail: gelsisy@yahoo.com