Freezability of buffalo semen by using tris extender supplemented with different concentrations of LDL

Shahba, M.I.¹, El-Sheshtawy, R.I.², El-Azab, A.I.³

1. Introduction

Artificial insemination (AI) is widely extended, for genetic improvement, after banking of the frozen semen [1].However, the genetic impact in AI industry is limited by the efficiency of semen production.

About 40-50 % of viable sperm lose their functional integrity during the freezingthawing process [2, 3]. The cryo-damage induced by the cryopreservation could be minimized and significantly improved by inventing the optimal cooling rate and efficient cryoprotectant [4, 5]. It has been © reported that the low density lipoprotein (LDL) is protective against cold shock, and hence, improves the sperm motility, acrosome and plasma membrane integrity as well as better protection against the sperm DNA fragmentation[6-9]. LDL has a cryo-protective property by adhering to the sperm cell membrane [10-12] and reduces the sperm membrane modifications [6,12] during the freezing-thawing process.

Although cryopreservation of buffalo spermatozoa has been performed routinely,

frozen spermatozoa after thawing at 37

Values within the same column with different letters differed significantly at least at P < 0.05 improvement of the existing cryopreservation protocols for buffalo spermatozoa is still targeted. The present study aimed at displaying the effect of LDL, in buffalo frozen semen extender, on the post-thaw sperm motility, sperm livability, acrosome and sperm cell membrane and DNA integrities.

2. Materials and Methods

Semen was collected from five mature buffalo-bulls by using an artificial vagina in Abbasia Frozen Semen Center, General Organization for Veterinary Services, Ministry of agriculture, Egypt. Immediately after collection, semen samples were kept in a water bath at 37° C for at least 5 min before evaluation. Only semen samples with at least 70% initial sperm motility and 80% normal sperm would be used for freezing.

For extension, Tris-Citric acid-Fructose-Egg yolk extender [13] was used as a control.

Instead of Egg yolk, LDL fraction extracted from the Egg yolk [6], would be added to the extender at6, 8, 10, and 12 %providing a concentration of 60 million sperm/ml. After freezing and thawing of semen in water bath at 37°C for 30 sec, evaluation of the sperm motility, livability and abnormalities

content Motility Livability Abnormality a a Egg yolk 31.50±2.36 71.80±1.65 10.00± 1.00 6% 29.50±1.90a 69.80±1.51a 13.20± 1.20 a a 8% 28.50±1.50 70.10±2.57 15.80 ±0.82 a a 10% 31.00±2.08 69.40±1.63 a a 12% 32.50±1.50 72.00±1.82 [14],the sperm membrane integrity by using the HOST [15] and the sperm DNA integrity by using the Acridine orange stain [16] were adopted as described by Shahba[17]. The obtained data were tabulated and computed for statistical analysis by SPSS® program, version 10 (1999) to assess, where appropriate, Mean ± SEM, ANOVA and LSD for the different sperm parameters at different concentrations of LDL when compared to those in the control.

3. Results and Discussion

Results

As shown table 1, with the exception of the sperm abnormalities, the present results did not show any significant difference in the post-thaw sperm motility and sperm livability as well as the sperm cell membrane and DNA integrities. The best semen quality was observed with the addition of 12% LDL to the frozen semen extender, as expressed by the highest sperm motility (32.50 ± 1.50 %), sperm livability (72.00± 1.82 %), sperm cell membrane (69.10 ±1.84 %) and DNA (98.90 ±0.91 %) integrities as well as the lowest sperm abnormalities (09.10 ±1.48 %).

C for 30 sec.

membrane integrity DNA integrity b a a 65.20 ±1.92 98.40 ± 0.73 a 66.10 ±2.00a 97.70 ± 1.00a a a a 64.10 ±2.58 97.90 ± 0.68 b a 11.60 ±1.40 65.50 ±2.63a 97.20 ±0.68 b a a 09.10 ±1.48 69.10 ±1.84 98.90 ±0.91

Discussion

LDL has been approved to be the effective part of egg yolk in cryopreservation of semen [8,18-20].The present study revealed that LDL addition to buffalo frozen semen extender offered slight better protection than egg yolk, a finding which came in agreement with some previous reports [6,7, 9, 21] indicating that LDL possesses remarkable cryopreservative properties. This effect was clear, especially when added at a concentration of 12% as expressed in terms of the sperm motility, livability, and abnormalities as well as the sperm cell membrane and DNA integrities. However, Munoz et al. [22] and Amirat-Briand et al. [23] noticed that the use of 8% LDL in Tris extender was more effective in the preservation of motility and plasma membrane integrity of bull spermatozoa. It has been suggested that LDL promotes incorporation of phospholipids and cholesterol into sperm membrane[12] and building a complex with seminal plasma proteins and making them unavailable to function in the cell membrane [12,24].

From the present study, the use of LDL at a concentration of 12% in buffalo frozen semen extender might be recommended to have better sperm motility, sperm livability, and sperm abnormalities as well as sperm cell membrane and DNA integrities.

References

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