Cryopreservation of South African indigenous ram semen
Munyai, Pfananani Hendrick
MetadataShow full item record
Semen was collected from the indigenous Damara, Namaqua Afrikaner, Pedi and Zulu rams. Hundred and twenty eight (128) ejaculates were collected throughout the entire study, with semen being collected twice a week (every Monday and Tuesday) from each ram, using the electro-ejaculator. Ejaculates were collected in graduated test tubes, placed in a thermo flask at 37°C, and transported to the laboratory for evaluation within 1h interval. The raw or fresh undiluted semen was then microscopically evaluated for volume, concentration, pH and sperm motility. The sperm concentration was determined with the aid of a spectrophotometer (Spermacue®) and the semen pH using a pH meter (Microprocessor pH/mV/°C Meter Hanna HI 931401). A Computer Assisted Sperm Analysis (CASA) system was used to evaluate the different sperm motility characteristics. All data were analysed using the statistical GenStat® program. The analysis of variance (ANOVA) was used to test for significant differences between treatments. Characterization of the South African indigenous ram sperm viability (percentage live/dead) of the semen samples was determined, using an eosin/nigrosin stain (60μl eosin/nigrosin and 6μl semen), in a thin smear. All sperm cells were evaluated on the same day of semen collection with the aid of a fluorescent microscope (BX 51TF), using an oil immersion objective (X100 magnification). The live sperm fluoresced green, while the dead cells stained red. The live sperm cells were further categorized as morphologically normal or abnormal. The volume of the indigenous ram ejaculates ranged between 0.4 and 0.9mL. The sperm concentration recorded in this study ranged between 0.9 and 1.3x109 sperm/mL, which are much lower when compared to other studies. The semen pH recorded in this study ranged between 6.5 and 7.3 and the sperm abnormalities ranged between 5.2% and 8.2% – which is regarded as acceptable for fertilization. To test the effect of storage temperatures on the viability of the diluted ram semen stored for different periods of time, the same procedure of semen collection and semen evaluation was followed. After the initial semen evaluation, all semen samples were pooled and diluted equally in an egg yolk citrate extender in the ratio of 1:1(v/v). The pooled semen sample was then divided into two portions, one sample being stored at 5ºC, and the other at 15°C, following storage periods of 3, 6, 9, and 24h respectively. Sperm characteristics were then recorded for each interval of storage. In general the percentage total motile sperm recorded after a 24h period of storage at 15°C was higher (61.2%), compared to that at 3h (51.4%), 6h (50.1%) and 9h (50.6%). From the results of this study it was concluded that diluted ram semen can be successfully stored for 24h at 15°C, retaining sperm motility for the application of AI. When evaluating the effect of glycerol as a cryoprotectant, in the diluted ram semen stored at two temperatures for different periods of time, the same procedure for semen collection and evaluation was followed. After initial evaluation, all semen samples were pooled and diluted equally with an egg yolk citrate extender containing 14% glycerol in the ratio of 1:1 (v/v), resulting in a final glycerol concentration of 7%. The pooled semen sample was then divided into two portions, one sample being stored at 5ºC and the other at 15°C, for periods of 3, 6, 9, and 24h. Sperm characteristics were recorded at each interval of semen storage. Semen stored at 15°C recorded a 48.3% total motile sperm after 3h of storage, but this increased to 50.4% following 24h of storage. The percentage of total motile sperm remained relatively constant at 40% after 3h of storage and 40.8% after 24h in the semen stored at 5°C. The addition of glycerol as a cryoprotectant demonstrated a protective effect on the sperm motility characteristics of sperm stored at both 5°C and 15°C for up to 24h of storage. The effect of different glycerol inclusion levels in the diluent, on the indigenous ram semen characteristics following cryopreservation were evaluated. The same procedure for semen collection was followed and semen was subjected to the initial evaluation comprising sperm concentration, semen pH and sperm motility. After initial evaluation of the ejaculates, the semen samples were diluted with an egg yolk citrate extender (EYC) fraction A (without glycerol), in the ratio of 1:1 (v/v) and cooled over a period of 2h to 5°C. All ram ejaculates were pooled and then divided into 4 portions treatment (groups). The first group was diluted with EYC (fraction A), which served as a control and the other 3 groups with EYC (fraction B) contained 7, 10 or 14% glycerol (GLY) in the ratio of 2:1 (v/v), making final glycerol concentrations of 2.3, 3.3 or 4.7% respectively. The semen samples were equilibrated for 2h and then loaded into 0.25mL semen straws. The straws were frozen in liquid nitrogen (LN2) vapour, whereafter semen straws were plunged into the LN2 (-196°C). The semen straws were thawed 7 days later, in a water bath (37°C) for 30 seconds. The sperm characteristics (motility and velocity) were microscopically evaluated using the Sperm Class Analyzer® (CASA) system. A 10% glycerol inclusion rate recorded a higher percentage of total motile sperm (15.6%), compared to the 7% glycerol (12.8%) and 14% glycerol (8.5%) inclusion levels, although all these differences were not significant. This study demonstrated that an egg yolk- citrate extender containing 10% glycerol can be used to cryopreserve indigenous ram semen effectively, based on the sperm motility characteristics. The low sperm motility results recorded when semen was cryopreserved in an extender containing 14% glycerol also indicated a degree of toxicity of glycerol at high inclusion levels in the semen extender. Regarding the conventional slow cryopreservation (programmable freezer) of ram semen versus semen cryopreservation in liquid nitrogen vapour, the same procedure for semen collection and evaluation was followed. After the initial evaluation of the raw semen samples, all ejaculates were pooled and then diluted using an egg yolk - citrate extender (EYC) fraction A (without glycerol), in the ratio of 1:1(v/v) and cooled over a 2h period at 5°C. After equilibration, the pooled semen sample was further diluted with EYC fraction B, containing 14% glycerol, in a ratio of 2:1(v/v)resulting in a final glycerol concentration of 4.7%. The pooled semen sample was then further equilibrated and loaded into 0.25mL semen straws. Half of the straws were frozen in liquid nitrogen (LN2) vapour and then plunged into the LN2. The other half of the semen straws were frozen with the aid of a programmable freezer. After 7 days, the semen straws were thawed in a water bath at 37°C, for 30 seconds. The sperm characteristics (sperm motility and velocity) were microscopically evaluated using the CASA system. From the findings in this study, it can be concluded that a controlled rate of semen cooling gave superior sperm motility results (15.3±3.0%), compared to semen frozen in LN2 vapour (8.8±0.9%). It should be noted that programmable freezers are costly, when compared to the liquid nitrogen vapour technique. Due to the fact that sperm motility differences recorded were not significant, it is suggested that the freezing of semen on a small scale be done using the LN2 vapour technique, without any significant decrease in sperm motility or possible fertility.