Modification of Extender and Cryoprotective Agents on Semen Preservation for Artificial Insemination Program in Horse
Abstract
A series of experiment had been conducted in several places i.e : local slaughter house of the district of Bantul Yogyakarta; Athena Stable, Cinere-Depok, Reproductive Rehabilitation Unit (URR), Faculty of Veterinary Medicine, IPB, Cavalery Stable of Parongpong-Lembang, Bandung and Artificial Insemination Centre of Ungaran, Central Java for two consecutive years, from 2004-2006. The objectives of this experiments were to study the artificial insemination program, including data base of anatomy and biometry of reproductive organ from Indonesian local horse, centrifugation technique and seminal plasma level, semen preservation and cryopreservation, synchronization of estrus and ovulation and insemination using liquid and frozen semen. The experiments consisted of five continous steps, accordingly. Results of the experiment I, indicated that the average weight and the thickness of the mare’s ovary was 22.9±8.2 g and 2.5±0.5 cm, respectively. The length of the oviduct was 22.7±1.2 cm, with the lenght and external diameter of uterine horns was 7.4±1.9 and 3.1±0.3 cm, respectively. The lenght and the diameter of the uterine body was 14.8±0.7 and 4.7±0.8 cm, respectively. The cervix was 8.0±1.0 cm long and 3.8±0.6 cm in diameter, while the lenght of the vagina was 23.5±3.4 cm. The length, circumference and thickness of the testis was 8.7±1.4; 12.5±2.4 and 3.8±0.9 cm, respectively. The testis volume and weight was 104.0±31.6 cm3 and 85.9±24.8 g, respectively. The length of epididymis was 10.6±1.5 cm. The lenght and the transversal section of bulbourethral glands was 5.0±1.4 and 2.9±1.5 cm with the prostate gland was 2.7±0.8 cm in long. The lenght and diameter of vesicular glands was 7.6±2.7 and 2.4±2.3 cm, respectively. The ampulla glands was 13.0±3.3 in long and 1.1±0.4 cm in diameter. The lenght and diameter of the penis was 48.8±4.5 and 3.4±0.5 cm, respectively and the length of glans penis was 5.5±1.7cm. In general the reproductive organs of the local horses were smaller size and weight if compared to the European type. The seminal plasma of stallion semen consists of high content of sodium chloride, which seems detrimental to the sperm preservation and cryopreservation. The seminal plasma should be removed by centrifugation technique to collect the rich sperm fraction only. The speed and duration of centrifugation affects the semen quality. In this experiment the semen were centrifuged at 2000 and 3000 rpm for 15 and 20 minutes. The level of seminal plasma content was investigates. Results of this experiment showed that the speed and duration of centrifugation had no effect on the quality of stallion liquid semen. The liquid semen consist of no seminal plasma showed 47.5% sperm motility and 61.0% of viable sperm which is significantly higher (P<0.05) compared of those with seminal plasma. It is concluded that the speed and duration of centrifugation had no effect on the semen quality while the removal of seminal plasma has beneficial for stallion sperm. The sperm metabolism during preservation at 5 oC decreased until 10% if compare to of those stored at room temperature. Sperm need carbohydrate such as glucose and fructose to support their live and motility. Semen stored at 5 oC caused chilling injury and affected there membrane plasma stability. Trehalose and raffinose play a role on membrane stabilization. The experiment was conducted to evaluate the effect of carbohydrates suplementation in skim and DV extender on the motility and viability of stallion sperm, stored in 5 oC and room temperature (24- 29 oC) respectively. Result of the experiment showed that the sperm motility and viability in DV extender stored at 5 oC were 40.0 and 65.6% higher (P<0.05) than of those stored in skim extender (31.1 and 44.3%), as well as in room temperature which were 38.0;55.0% for DV and 28.0; 50.8% for skim extender respectively. The sperm motility and viability of extender supplemented with fructose at both temperature showed (40.9 and 37.7%) higher percentage if compare to trehalose (34.4; 32.7%), raffinose (34.6; 31.2%) or control group (32.2; 31.0%). The best extender and carbohydrats combination was DV fructose with the total sperm motility stored at 5 oC for 96 h observation was 45.1% higher then DV trehalose (40.2%) and DV raffinose (39.2%). The sperm motility in DV control and skim fructose extender was 35.3 and 35.6% higher then skim control (28.9%); skim trehalose (30.3%) and skim raffinose (29.6%) extender. The result concluded that the suplementation of fructose in DV extender was the best combination to maintain stallion sperm motility and viability compare to the other combination. Cryoprotective agents (CPAs), protect the sperm during cryopreservation. There are two general classes of CPAs, at first penetrating cryoprotectants, these pass through the sperm membrane and act both intracellular and extracellularly, and the second non-penetrating cryoprotectants, these act only extracellularly. Intracellular cryoprotectants such as glycerol are beneficial due to its function to lower the freezing point. Non-penetrating cryprotectants include proteins as been proved in milk or egg yolk; sugars such as fructose, lactose, mannose, raffinose or trehalose; non-penetrating CPAs move water out of the sperm which results in dehydration and shrinkage. The first experiment was the supplementation of different carbohydrates in skim milk extender supplemented with 50 mM trehalose, 50 mM raffinose and 100 mM fructose, with the concentration of sperm 200x106 mL- 1 . The second experiment was conducted with the addition of different CPAs namely glycerol (G), combination of ethylene glycol with glycerol (EG) and dimethilformamide (DMF) using two different extenders i.e. skim milk and Dimitropoulos (DV). Results of this experiment indicates that there were no significant difference (P>0.05) in frozen semen quality using different carbohydrates supplementation. Dimethilformamide was the best CPA compared to the others; there were no significant difference on the percentage of motile and viable sperm in DV and skim milk extender. This Study confirmed that DMF in DV extender was the best combination. The last experiment was the used of preseved semen to inseminate the estrous mares. Nineteen and twelve mares were used in the first and second group of inseminations. At the first attempt, the mares were synchronized with double injection of PGF2α at 14 days apart. The follicle size were monitored using ultrasound scanner during the third day of estrus, and 2500 IU hCG was administered at the same time. The AI was conducted 35 hours after hCG with total motile sperm 200x106 for liquid semen and 250-300x106 for frozen semen. At the second group, the mares were synchronized with single injection of PGF2α. The follicle size were monitored on the third day of estrous, and 2500 IU hCG was administered at the same time. The follicle size was monitored on 24 and 30 hours after hCG injection. The insemination were conducted when the follicle reach the stage of preovulatory size commonly known as “pear shaped” with total motile of sperm 200x106 for liquid semen and 350-400x106 for frozen semen. The response of the estrous with double injection of PGF2α was 73.7% and lower than of these with single injection (83.3%). The conception rate (CR) of the first group was 14.3% with frozen semen and 42.9% using liquid semen while in the second program the CR was 66.7% with frozen semen and 50.0% using liquid semen. The average of pregnancy rate was 45.5% with frozen semen and 38.46% with liquid semen. The average of pregnancy rate in both semen was 41.7%. In conclution, the administration of single PGF2α in mare have a responsive CL for estrus synchonization was more efective than double PGF2α. The total sperm motile 350-400x106 for frozen semen give better result than 250-300x106 and the follicle size and activity should be monitor after hCG administration periodictly.