Cryopreservation of Buck Etawah Crossbreed Semen Using Tris-egg yolk and Tris-soya Diluents with Carbohydrate Modified and Different Cryoprotectant

Abstract

Artificial Insemination (AI) is known as a technology to improve the genetic quality of goats in Indonesia. AI in goat is still limited in its practical use; this is related to a variety of constraints, which one of those would be the guarded quality of frozen semen. The quality of frozen semen is determined by various factors, including the freezing techniques, the diluents and cryoprotectants. Buck frozen semen produced by AI centers usually use Tris-egg yolk (TEY) diluent. Egg yolk of animal origin has been recognized as a highly potential media allowing transfer microorganism to female tract through AI. Therefore, some research studies have been focusing to find another diluents using soy extract as an alternative of diluent. Preservation of buck semen using soy extract contains plant phospolipid has not been well studied. Dissacharide (trehalose) or trissacharide (raffinose) acts as an energy source as well as an extracellular cryoprotectants in diluents of frozen semen; these would aid in protecting the sperm during freezing process. Dimethylformamide (DMF) has smaller molecules than glycerol may act as a beneficial cryoprotectant when it is added into the diluents, showing a better effect in protecting the sperm membrane during freezing. This study was conducted in two steps: 1) Preserved liquid semen using tris-egg yolk (TEY) and tris-soya (TS) diluents with either trehalose and raffinose supplementation; 2) Produced frozen semen using TEY and TS with supplementation of the combination showing the best results in step 1. Both diluents used DMF and glycerol as the cryoprotectants. Semen were collected from three sexually mature bucks using artificial vagina, evaluated and divided into six aliquot treatment groups. Each of aliquot diluted with TEY and TS supplemented with 50 mM trehalose or raffinose, which contain 50x106 sperm concentration per 0.2 mL volume. All of them kept in refrigerator in 5°C. The progressive motility, viability and plasma membrane intact (PMI) were evaluated every 12 hours up to the time when only 50% of sperm display motility. The combination of trehalose or raffinose based showing the best results in analysis during the step 1 was added with TEY and TS diluents, supplemented with glycerol and DMF cryoprotectants in frozen semen process. Extended semen were kept in minitub straw, containing 100x106 sperm/0.25 mL. Equilibration time was 4 hours in 5°C, then freeze in N2 vaporated for 10 minutes in styrofoam box. The result showed that semen quality from three bucks were good, such as the average of volume was 1.29±0.21 mL, creamy and thick in consistency. The concentration was 3608.33±657.70x106/mL, motility was 77.78±2.56%, viability was 85.13±4.38%, PMI was 77.72±3.52% and abnormality was 7.51±2.88%. The motility of sperm kept in liquid semen diluents, TEY supplemented with trehalose or raffinose gave better result, it can be maintained sperm motility up to 50% for 72-84 hours, compared to TS diluent (48-60 hours) (P<0.05). TEY diluent supplemented with trehalose can maintained sperm motility longer (52.82±3.21%) for 84 hours, compared to raffinose supplementation (51.78±4.41%) and control (51.11±4.86%) for 72 hours. The viability of sperm evaluation showed 6-9% higher than sperm motility percentage, while the percentage of PMI gave similar percentage as sperm motility. The quality of semen after dilution were similar among treatments compared with fresh semen quality and there were no differences between treatments (P>0.05). However, the influence of equlibration process for 4 hours kept in refrigerator slightly decrease to sperm motility (1.69%-2.24%) but there were no difference between treatments (P>0.05). The quality of semen post thawed, sperm motility were decreased in TEY trehalose added glycerol and DMF cryoprotectants (11.60% and 15.00%) better than in TS raffinose with similar cryoprotectants (34.45% and 37.60%) (P<0.05) and there were difference between TEY trehalose and TS raffinose added DMF and glycerol cryoprotectants (P<0.05). The sperm motility after thawed using TEY trehalose added glycerol cryoprotectant showed higher percentage (65.07±5.38%) with recovery rate (RR) 83.65% compared TEY trehalose added DMF (61.67±5.55%) with RR 79.29%. In contrast result, sperm in TS raffinose with DMF, can maintain sperm motility better (42.22±8.13%) than added with glycerol cryoprotectant (39.07±5.38%) (P<0.05). In conclusion, Tris-soya can be use as a liquid diluents for buck semen and supplemented raffinose can maintained viability of sperm up to 60 hours, whereas the supplemented of trehalose to TEY diluent gave higher sperm motility, viability and PMI up to 84 hours. Glycerol cryoprotectant added to TEY diluents showed a better role to protected sperm in frozen semen process, compared TS diluents with DMF.