The goal of this study was to generate a database of laboratory results for African lion (Panthera leo) blood to obtain reliable reference ranges to augment what is currently available in literature. Also to investigate the possibility of age and sex having an influence on these reference ranges. The specific objectives of this study were: o to determine reference values for haematological and biochemical blood variables for lions bred in captivity, as a function of age and sex; o to evaluate the Beckman Coulter Acâ¢T 5diff Haematology Analyzer for lion differential white blood cell analyses; o to determine morphometric measurements and establish reference growth curves (and range reference values) for lions bred and reared in captivity as a function of age and sex; o to determine reference values for some practical and meaningful body measurements and their correlations. This study was conducted on three lion ranches in the Free State province and at the Bloemfontein Zoological Gardens (Bloemfontein Zoo) with captive lions (Panthera leo) of both sexes and ages ranging from three months to nine years. Lions were divided into four age groups according to published literature. Animals were chemically immobilized (darted) with Zoletil® 100 at 4 to 5mg/kg in their holding camps and moved to a shaded place as soon as the drug had taken its full effect. Blood was collected into three different types of blood collection tubes and body measurements were taken. This was all done as fast as possible before the effect of the immobilizing drug could wear off. In some cases it was necessary to give an animal a top-up dose to prevent it from waking up too quickly. Animals were moved back to their holding camps to fully recover from the immobilization. Blood analyses done with the Acâ¢T 5diff Haematology Analyzer from Beckman Coulter® for haematological parameters was conducted within 30 minutes after blood collection. Blood for biochemistry parameters was centrifuged, serum collected and cryo preserved at -20°C until it could be taken to the laboratory for analyses. Blood smears were made on the lion ranches and Bloemfontein zoo immediately after the analysis with the Acâ¢T 5diff Haematology Analyzer, fixed and packed for transport to the laboratory. At the laboratory the serum was used for biochemistry analyses, using standard laboratory techniques. Blood smears were stained and examined under a light microscope for the differential white blood cell count by means of the manual-visual method. Results were statistically analyzed to determine reference ranges and the influence of age and sex on these reference range values for the different parameters, were considered. Body measurement were also statistically analyzed to determine correlations between body weight and different other measurements. These correlations were then used to determine if it will be possible in a field situation to use the age and sex of an animal together with a certain body measurement to estimate body weight accurately, if actual weighing was not possible. From these analyses it was concluded that age and sex do have an influence on blood analysis and blood reference ranges for the African lion (Panthera leo). Unfortunately, it differs between parameters and there is not one rule to apply. The conclusion could also be made that body weight could be determined by measuring the head length of an animal. More research is warranted to obtain more data set and establish range reference values that can be validated and used with a high degree of confidence in the lion breeding industry.

In the first study performance data from 465 Boer goat rams tested in a central performance test in the Northern Cape Veld-Ram Club from 1989 to 2007 were analysed to determine the relationship between performance and sale price. Rams were subjected to extensive management conditions on natural pastures for 160 days and finished-off in a feedlot for 50 days. Upon the conclusion of the entire test period, the rams were auctioned. Performance information was available for buyers for decision making. Traits analysed included final weight (FW), final weight index (FWI), average daily gain (ADG), average daily gain index (ADGI), growth per day of age index (ADOI), Kleiber ratio (KR), auction weight (AW), scrotal circumference (SC), selection index (SI) and sale price (SP). Stepwise regression analyses, using proc GLM of SAS were performed to identify variables that significantly influenced sale prices. Final weight was significant in eight out of eighteen years, auction weight was significant in six of the seven years measured and selection index influenced prices in seven out of ten years. Scrotal circumference, average daily gain, final weight index, average daily gain index and Kleiber ratio had little influence on sale price. Growth per day of age index did not show any influence on sale price. Price was positively correlated (P<0.05) with many of the performance traits. All significant correlations were moderate to high and ranged from 0.37 to 0.80. The amount of variation in sale price accounted for by the performance traits ranged from 15% in year 1991 to 65% in 1998. The most important traits influencing sale price (SP) were final weight, auction weight, and selection index. The results indicated that buyers of stud rams put more emphasis on production traits such as body weight and that they recognize the importance of performance data as shown by their preference for animals with high selection indices. In the second study data consisting of 3855 records and collected from 1998 to 2008 were analysed to estimate genetic parameters for economic traits in two Boer goat flocks. The traits investigated were weaning weight and post-weaning weight. Least square analysis was used for estimation of environmental effects. Genetic parameters were estimated from single and bivariate trait analyses using ASREML software fitting animal models. By ignoring or including maternal additive genetic effects and their covariance and maternal permanent environmental effects seven different models were fitted for each trait. The fixed effects of sex, type of birth, age of dam, year of birth, herd, season and age of lamb, were all significant (P<0.05) for both traits. The direct heritability estimates varied from 0.24 for weaning weight to 0.31 for post-weaning weight. The corresponding maternal permanent environment due to the dam was 0.10 and 0.44 respectively. The maternal heritability (0.03) for weaning weight was lower than its corresponding direct heritability. Estimates of genetic parameters in this study confirmed that selection for weaning weight would result in genetic improvement of Boer goats.

The main aim of this study was to evaluate the effectiveness of repeated inoculations of a Corynebacterium cutis lysate (Ultra-Corn®) - a non-specific immune-stimulant, to reduce the milk SCC in commercial dairy cows. An additional aim was to evaluate if these inoculations had any detrimental effects on milk quality. This study was performed in two separate trials, using Holstein cows with SCCâs over 250 000 cells/ml of milk at different stages of lactation from two commercial dairy farms in the Free State Province. On each farm, cows were paired according days in milk and SCC, in order to obtain two homogeneous groups of experimental animals. The two groups of cows in each farm were randomly allocated to a treatment or a control group. Both groups in the same farm were managed under the same conditions for the entire trial periods. The only difference was that cows from the treatment group received 3 weekly inoculations of Corynebacterium cutis lysate(Ultra-Corn® ), while those from the control group received distilled water for injection (the same volume as the cows in the treatment group). Two similar trials were conducted, using the same basic experimental design. Differences were only in the dose of the Corynebacterium cutis lysate inoculated per cow treated, number of experimental animals and duration of the observation periods. In Trial 1, cows from the treated group received 3 weekly vaccinations of Ultra-Corn® (4 ml per cow, thus 80mg of Corynebacterium cutis lysate per cow) injected subcutaneously (sc), while those from the control group received 3 weekly sc injections of 4 ml distilled water. This was followed by 8 weeks of observation of the effect of treatment on milk SCC and composition. In Trial 2, the three doses of Corynebacterium cutis lysate administered weekly per cow for the treated group was 2ml/100kg, thus 40mg Corynebacterium cutis lysate/100 kg per cow. This was followed by 8 weeks of observation of the effect of treatment on milk SCC and composition. Individual quarter milk samples were collected weekly from all cows and analysed for SCC and a combined milk sample (from the measuring bottle in the milk parlour) from each cow was also taken for butterfat, protein, lactose and urea content. The results were compared between the two groups per farm, using ANOVA procedures for repeated measures analysis, using the 95% confidence level (SAS, 2004). The two farms were evaluated separately, due to the possible differences between general management conditions, which could introduce serious confounding factors if the results from the two farms were combined. However, it can be considered that both dairy farms used an acceptable level of commercial dairy management practices and produced an acceptable yield per cow under South African commercial conditions. In general no significant differences were recorded between the treated and control groups of cows in both farms in both trials in terms of milk SCC, butterfat, protein, lactose and urea content of the milk. In this study, the immuno-stimulant effect of Ultra-Corn®, a Corynebacterium cutis lysate could not be confirmed in lactating cows. Although this inoculant does not seem to have any detrimental effects on the main solids of the milk, its use cannot be justified as it did not significantly reduce somatic cell counts in lactating cows. Further research is warranted to develop and evaluate the effectiveness of vaccines against mastitis causing organisms, in order to control SCC and mastitis in dairy cows. However, when such studies are conducted it is advisable to use very high number of experimental units and proper control trials should be conducted. All efforts should be done to ensure minimum environmental changes during these trials, which can introduce serious confounding effects in the experimental design.

The aim of the study was to characterize and evaluate the quality of fresh semen of 4 breeds of chicken and the susceptibility of cockerel semen to a cryopreservation protocol, assessed microscopically for sperm motility and morphology and ultimately fertilizing ability following AI. The differences between breeds were determined by comparing the fertilizing ability and hatchability of fresh and frozen-thawed semen. The study was carried out at Glen Agricultural Development Institute and at the University of the Free State. Four chicken breeds, namely the Rhode Island Red, Potchefstroom Koekoek, New Hampshire and White Leghorn, were used. Qualitative characterization of the semen was performed in 28 cockerels (7 per breed). Semen was collected using the massage technique twice a week in the first trial. The eosin-nigrosin staining technique was used to microscopically evaluate the morphology of the sperm from the different breeds. The fresh semen parameters evaluated were ejaculate volume, semen colour, semen pH, sperm concentration, the percentage live and dead sperm, sperm motility and the abnormalities of the sperm. The percentage live and dead sperm, sperm motility and abnormalities were also evaluated for the frozen-thawed cockerel semen. During the second phase of the study, semen was collected 3 times per week from the same cockerels. Semen was frozen using a fastâfreezing procedure on dry ice, with 10% DMSO as the cryoprotectant. AI was performed on 4 different breeds of hens (20 hens per breed) (Rhode Island Red, Potchefstroom Koekoek, New Hampshire and White Leghorn), using fresh semen (control) and frozen-thawed semen. During AI of each breed, 10 hens were inseminated with fresh and the remaining 10 hens with frozen-thawed semen. The sperm characteristics of the semen samples of the 4 breeds recorded were ejaculate volume, ranging from 0.3±0.1 to 0.4±0.1ml, semen pH of 7.6±0.4 to 7.7±0.3, sperm motility (scale of 0-5) 2.8±0.8 to 3.1±0.9, estimated sperm motility 58.8±12.5 to 63.8±13.6%, ejaculate concentration (x109 sperm/ml) of 320.4±286.5 to 748.5±475.3, percentage live sperm 75.6±29.1 to 81.5±26.8%, and the percentage dead sperm 18.6±26.8 to 24.4±29.1% respectively, with the percentage normal sperm ranging between 77.3±17.1 and 84.8±9.0%. Head, mid-piece, tail and other sperm abnormalities of the fresh semen of the 4 breeds ranged from 2.9±3.3 to 7.7±9.6%, 7.9±5.2 to 11.0±7.0%, 0.4±0.8 to 1.9±3.0% and 0.6±0.9 to 1.5±1.9%, respectively. Semen samples were frozen in pellet form on a block of dry ice, by pipetting into the indentations on the surface of the ice. The frozen cockerel pellets were thawed following cryopreservation, by being placed into a test tube in a water bath (60°C), and the tube shaken continuously until complete thawing of the pellet. During the time of semen cryopreservation, a decrease in the number of live, morphologically normal sperm, and increase in the percentage dead sperm and sperm with abnormalities were recorded. The freeze-thawing process caused a significant (P<0.05) decrease in the percentage live sperm and the sperm motility, ranging between 37.4±10.4 and 42.3±12.1% and 3.6±0.5 and 3.9±0.3 respectively. A consequent increase in the percentage of dead sperm (between 57.7±12.1 and 62.4±10.8%) was also recorded. The sperm abnormalities regarding sperm head abnormalities ranged between 17.3±3.8 to 22.5±10.3%, the mid-piece abnormalities 7.9±3.8 to 10.4±2.0% and the tail abnormalities between 0.5±0.9 to 2.0±2.4% respectively for the thawed semen. Frozen-thawed semen was thawed in a water-bath 60°C and hens were inseminated twice per week using the frozen-thawed semen, and once a week with fresh semen for a total period of two weeks. Data for the two trials were analyzed using the ANOVA and the Tukeyâs Studentized Range (HSD) test for repeated measures (SAS system General Linear Models Procedure). A total of 973 eggs, from all breeds of chicken namely the Rhode Island Red, Potchefstroom Koekoek, New Hampshire and White Leghorn were collected following AI with fresh and frozen semen from individually caged hens. Eggs were collected, incubated and hatched to check if fertile and normal chicks could be produced from frozen-thawed cockerel semen. The difference in fertility and hatchability of the hens of the different breeds were compared and found to be highest in Rhode Island Red, White Leghorn, and Potchefstroom Koekoek respectively and lowest in New Hampshire using fresh semen. When using frozen-thawed semen, the sequence of fertility performance was the White Leghorn, Rhode Island Red, Potchefstroom Koekoek and New Hampshire, respectively. The effect of the numbers of sperm per AI dose on fertility, age at embryonic death, and hatchability of fertile eggs were also evaluated. Low numbers of sperm per AI in the hens resulted in a decrease in the total number of chicks hatched. The lowest fertility rate recorded was in the New Hampshire (2.7%), when using frozen-thawed semen to inseminate the hens. This may be attributed to the low numbers of sperm inseminated per AI dose. Egg hatchability of the fertile eggs was high in the White Leghorn (13.6%), Rhode Island Red (12.8 %), Potchefstroom Koekoek (9.7 %) and low in New Hampshire (2.7%) respectively, which could possibly be attributed to the egg size. Medium sized eggs were preferable for setting, in order to obtain an acceptable hatch, as they generally hatch better than the larger eggs. The results recorded for fertility and hatchability in the control group (fresh semen), was similar to the results recorded by other researchers, showing that the AI method used was acceptable. There still exists a necessity to develop an ideal cryopreservation method (diluent and freezing procedure), which would allow for acceptable long term storage of cockerel semen in liquid nitrogen (-196°C) for future use and export with minimum loss regarding sperm viability and fertilizing capacity.

A laboratory study was under taken to investigate the nutritional value of different Opuntia varieties from chemical analysis. One year old cladodes from six different varieties of Opuntia ficus-indica namely Castello, Chicco, Fusicaulis, Montery, Morado and Rubasta were randomly harvested in five replicates. The highest (P<0.05) average dry matter (DM) content was observed for the Chicco variety and was the average for all varieties generally low (9.13%). There were no significant difference (P>0.05) in ash content. Significant (P<0.05)differences among varieties were recorded for crude protein (CP) (3.7 to 8.1%), acid detergent fibre (ADF) (13.6 to 17.4%), neutral detergent fibre (NDF) (19.9 to 38.5%), cellulose (2.4 to 14.8%), hemicellulose (4.5 to 12.7%), lignin (2.51 to 21.5%), non fibre carbohydrates (NFC) (33.4 to 48.6%), ether extract (EE) (1.9 to 2.4%). The average mineral composition were as follows, phosphorus (P) 0.18%, potassium (K) 3.02%, calcium (Ca) 2.3%, magnesium (Mg) 3.6% and sodium (Na) 0.04%. It was concluded that in general cladodes could be classified as a high moisture energy source with an energy level between that of roughages and concentrates with a low CP and high Mg, K and Ca content. In a second study the influence of dry matter (7.12 to 27.6 %) and molasses (0 to 24%) content on the fermentation characteristics of Fusicaulis cladode silage was investigated. One year old cladodes was ensiled in three litre square plastic bottles (six replicates). A higher DM content were characterized with a lower (P<0.05) ADF, NDF, CP and EE content. The inclusion of molasses resulted in a lower (P<0.05) ADF, NDF and EE content. An increased (P<0.05) acetic acid (AA) content in Fusicaulis silage was observed as the level of DM and molasses increased. A higher silage DM content resulted in a lower (P<0.05) propionic acid (PA) and butyric acid (BA) content. No significant (P>0.05) influence of molasses on PA and BA occurred. Lactic acid (LA) content and pH of cladode silage was increased (P<0.05) by higher DM and molasses levels. It was concluded that the content of cladode silage could have detrimental effect on intake and animal performance. In a third study, the effect of DM (35.5 and 37.4%) and molasses (0 and 24%) content of ensiled cladodes on dry matter intake (DMI) and apparent digestibility by sheep was investigated. Twentyfour merino wethers were randomly divided into 4 groups of 6 each. No statistical significant (P>0.05) influence of DM and molasses levels in Fusicaulis silage on the apparent digestibility of DM and CP as well as metabolizable energy (ME) content occurred. The inclusion of molasses resulted in a reduction in the ADF (P=0.08) and NDF (P=0.05) digestibilities. Apparent EE digestibility was reduced (P<0.05) by a higher DM level. DM content of cladode silage had no influence (P=0.42) on DMI by sheep. The inclusion of molasses influenced DMI favourably (P=0.54). In contrast with molasses, a higher DM level (37.4%) in silage resulted in a higher (P<0.05) metabolizable energy intake (MEI). Cladode silage supplied more or less in the ME requirements of 30 kg wether lambs. Weight losses were decreased (P=0.07) by the inclusion of 24% molasses. It was concluded that the laxative effect of cladode silage resulted in a higher rate of passage through the digestive tract. Accordingly the digestibility and ME content of cladode silage is relatively low. Accompanied by a low DMI, cladode silage as a sole energy source supplied only in the maintenance requirement of sheep, especially when molasses was included at ensiling.

Two trials were conducted at Agricultural Research Council (Irene â Pretoria), between March (autumn) and October (spring), 2008. The first trial evaluated the effect of the oocyte harvesting technique on the quantity and quality of ovine oocytes recovered, as well as the effect of the culture media used on embryonic development. The second trial evaluated the effect of breed and semen cryopreservation on the embryonic development following in vitro fertilization (IVF). For the first trial, ovine ovaries were collected from slaughtered animals at the Boekenhout abattoir near Pretoria. All ovaries were immediately transported to the laboratory for further processing and use in the trials. On reaching the laboratory, ovaries were processed, and oocytes were harvested, either by slicing (140 ovaries), or follicular aspiration (167 ovaries). The oocytes collected were matured, fertilized with fresh ram semen and cultured in order to produce embryos. The average total number of ovine oocytes recovered per ovary, and the total number of oocytes collected were higher (P<0.05) when using the slicing (5.2±0.4 oocytes/ovary and 75.3±12.9 total oocytes) technique, compared to the aspiration technique (2.6±0.5 oocytes/ovary and total of 40.3±9.1 oocytes). The number of acceptable quality (intact cumulus layers) oocytes was also higher (P<0.05) following the slicing (46.7±10.3 oocytes), compared to aspiration (10.3±2.0 oocytes) technique. However, the number of poor quality oocytes did not differ between the 2 oocyte harvesting techniques. The acceptable oocytes were then matured in vitro for 24h and no difference (P > 0.05) in oocyte maturation rate between the oocytes recovered using the slicing or aspiration technique was recorded. A comparison of the 3 culture media (Potassium simplex optimization medium - KSOM, Synthetic oviductal fluid - SOF and Charles Rosenkrans medium - CR1) used for maintaining subsequent embryonic development was then evaluated. All oocytes were further matured, using the maturation medium - TCM 199 containing FSH, LH and E2, supplemented with 10% FBS. After maturation, the oocytes were fertilized (using fresh ram semen) and incubated for a period of 18h. At the end of 18h fertilization period, oocytes were vortexed in an Eppendorf tube containing 100μl M199 + 10% FBS for 1.5min. The vortexing was performed to remove the cumulus cells surrounding the zygote. A total of 1405 presumptive zygotes were randomly allocated to the 3 different culture media (481, 461 and 463 zygotes in KSOM, SOF and CR1, respectively). The zygotes were then cultured for a period of 7 days. No significant difference between all 3 culture media was recorded regarding cleavage rates, showing that culture media had no effect on the subsequent cleavage. However, the percentages of embryos decreased with an advancement of the embryonic developmental stages.

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A sound breeding objective is the basis for genetic improvement in overall economic merit of animals. Breeding objectives for Holstein and Jersey dairy cattle breeds in South Africa were developed in the current study, using a systematic approach. First, a logical framework with a profit focus was utilised to develop plausible selection goals for the pasture-based and concentrate-fed dairy production systems in South Africa, leading to an exhaustive list of objective traits influencing these goals and subsequently their possible selection criteria. Next, economic values were calculated for those objective traits for which there was adequate bio-economic data, viz.: milk volume, fat yield, protein yield, live weight, longevity, calving interval and somatic cell score. A bioeconomic model, simulating typical South African pasture-based and concentrate-fed herds, was used to calculate economic values by determining changes in profit arising from an independent unit increase in each trait. Alternative payment systems of four major milk buyers in South Africa were used. Relative economic values, standardised to the value of protein, were used to compare the relative importance of traits across breeds, production systems and payment systems. Protein yield, fat yield and longevity consistently had positive economic values and the converse was true for body weight and calving interval. Economic value for volume was positive or negative, depending on whether the payment system paid for it or did not. Economic values were reasonably robust to fluctuations in the cost of feed and price of beef; with the exception of fat, whose value became negative beyond the feed price of ZAR3.50. Protein was, overall, the most important trait, although volume, live weight, longevity and somatic cell score were more important in some situations. Calving interval was the least important trait, its value ranging from 4 to 22% compared to that of protein, probably because the model used underestimated its value. Sire rankings on aggregate EBVs based on these economic values did not differ much across breeds, production systems and payment systems, most rank correlations falling in the range 0.70-0.99. A single breeding objective may therefore be used for both the Holstein and Jersey breeds, across the different production and payment systems. The basis for multiple-trait selection in the major cattle breeds in South Africa has thus been developed. Considerable work, however, needs to be done to enhance this breeding objective as well as facilitate its wide adoption by industry.

Wag-ân-Bietjie Private Nature Reserve is situated ±30 km north of Bloemfontein in the summer rainfall area. The northern part is 437 ha in size and represents a transition between grassland and riparian vegetation. Vegetation types present are grassland, open thickets, dense thicket, drainage lines of the Modder River, a wetland and disturbed area. Phenology (seasonal leaf carriage) of plants formed the basis of this study. Percentage leaves in each phenophase (Budding-, Immature-, Mature-, Yellow- and Dry leaves) was noted fortnightly for specific marked trees and shrubs representing each vegetation type. The deciduous nature of woody species influenced quality and quantity of browse available for herbivores. Consequently the nitrogen concentration in faeces (Nf) of four game species was determined to indicate their nutritional status through the different seasons. The rise and fall of Nf values corresponded to the seasonal increase and decrease of leaves (phenology pattern). Nf ranged during four years from 18 â 37 gN/kgDM for giraffe, 14 â 33 g/kg for kudu, 16 â 35 g/kg for eland and 17 â 28 g/kg for impala. Abovementioned minimum concentrations are close to, and in the case of kudu below known critical values where animals start to lose body condition. Nitrogen is the most limiting nutrient in the dry, cool season and is linked to protein percentage present in browse. Browse becomes a limited resource in the winter, therefore certain game species moved seasonally to different areas inside the private reserve in search of food. It was decided to supply feed in order to sustain animals and help them maintain body condition during the critical period that was established to be from July/August to middle October. The duration of feeding is important and it is recommended to start feeding from July at a low ration and then gradually increase feed towards the end of the critical period in correspondence with the declining browse and grass resources. Average monthly leaf carriage percentages were used to calculate browsing capacity per month in each vegetation type and in the study area as a whole. Browser units that could be sustained on browse resources within the 0 â 2 m stratum ranged from 1 â 6.7 BU between winter and summer due to the deciduousness of all woody plants present in the study area. This justifies in some way the provision of feed, or else the numbers of animals would need to be reduced to 1 BU which does not represent sustainable populations. Viable population numbers, economic value, diet and reproduction rates were used in determining the numbers of individual animals that can be stocked. Grazing capacity of the area differed according to annual rainfall and increased with higher rainfall. Consequently it needs to be recalculated annually. Habitat occupied by all 17 herbivore species was determined. Some species did not historically occur in the province. Most of them have adapted to the central Free State conditions over time, while others were introduced more recently. Inter-species competition for space and food resources proved to be high in the study area. A reduction in animal numbers has been recommended to limit competition. There is an ever increasing number of private game ranches in the province, >343 in August 2010, that will benefit from this research. Some general, operational guidelines have been presented that are applicable to the management of other game ranches in the province as well. When calculating individual animal numbers equivalent to carrying capacity values of other areas, the percentage grass and browse that herbivores include in their diet need to be adjusted to the specific area for accurate stocking densities.

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% â 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.