الفهرس 
Rumen Physiology and RuminationOnly 14 pages are availabe for public view
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Abstract
The aim of this work was : 1-To identify the different types of ciliate protozoa that are present most commonly in sheep rumen.2- To study the possibility of cryopreservation of rumen ciliates with establishment of the most suitable method for cryopreservation.3- To create a rumen protozoal bank that can be used for treatment of clinical cases of indigestion.
4-To conduct trials for using of the cryopreserved rumen ciliate protozoa to improve faunation in sheep after experimental defaunation.Therefore this work divided into two experiments. The first is the cryopreservation experiment and the second is the transfaunation (or refaunation) experiment.The trial is considered the first of its type in Egypt and in the Middle East. The experiment is done by slow interrupted freezing by using nitrogen vapor and liquid nitrogen.I- Cryopreservation Experiment :A-Sampling :For cryopreservation experiment, approximately 500 ml of ruminal contents was collected from slaughtered sheep or collected from live by stomach tube before their morning meal and transported to the laboratory at 39°C in anaerobic condition to be used for cryopreservation.B-Rumen protozoa count in rumen content :The total protozoal count was conducted. In this method, the usual slide and cover were used for direct microscopic count of protozoa by counting 0.1ml.of the diluted ruminal sample.
C- Equilibration of Protozoa with the Cryoprotectant Additive :
Equilibration of Protozoa with the cryoprotectant additive was conducted . In this method, the cryoprotectant (DMSO, glycerol, or ethylene glycol) was mixed with the ciliate suspension (with a minimum of 104 cells/ml) to obtain final concentrations of 4, 5 and 6% for each cryoprotectant followed by equilibration at 25°C in a water bath for 5 min. (equilibration time), and 0.2 ml of the mixture was placed in 0.5 ml straw before freezing D-The two-step cryopreservation of rumen ciliate protozoa : Cryopreservation was carried out by the two-step freezing method with little modification. Ten straws containing the mixture of ciliates and the cryoprotectant (10 straws for each cryoprotectant) were placed in a cooling water bath at 5°C for 30 minutes followed by holding the straws in nitrogen vapor (-25°C) for 45 minutes (holding time) then direct immersion in liquid nitrogen at -196 °C where straws remained there until the thawing step just prior to examination. E-Thawing of cryopreserved rumen protozoa :=Thawing of cryopreserved rumen protozoa was conducted . Frozen straws were removed from the liquid nitrogen vessel and placed in a water bath at 39°C for 5 min.=F-Evaluation of Survival Rate := The survival rate was evaluated . After thawing, the survival rate was estimated by counting the proportion of motile ciliate under a microscope monthly for 6 months.II Defaunation experiment : A- Experimental design :Nine female sheep with average live weight of 22kg were randomly divided into 3 groups:1-The first group included 3 sheep that were fed on basal diet and used as control. 2-The second group included 3 sheep that were fed on basal diet and single oil drench (cooking oil at 5ml / kg BW) for defaunation.3-The third group involves 3 sheep that were refaunated by intraruminal injection of cryopreserved ruminal protozoa. B- Defaunation by oil treatment :The defaunation was conducted . The animals were adapted to the diets for 2 weeks before giving the oil. Then 5 ml/kg live weight was administered directly into the rumen using a stomach tube after fasting of animals for 18 hours.C-Refaunation technique (in vivo inoculation of cryopreserved protozoa) :The refaunation was conducted . The animals in group 3, were refaunated by intraruminal injection of one cryo-straw with the 5% DMSO as cryoprotectant (for each sheep) after being thawed at 39 °C.D-Ruminal juice examination :1- Determination of ruminal pH :The pH of the collected rumen juice was determined by using a pH meter .2- Estimation of ruminal ammonia nitrogen concentration : The rumen ammonia nitrogen concentration was determined by distillation method .E-Determination of body weight and body weight gain : Sheep were weighed in the three groups (control, defaunated and refaunated) on day 0 (just before inoculation) and on days 30, 60, 90 and 120 post-inoculation. Then the live weight gain (BW) per month and per day was calculated F- Hematological examination :1- Blood samples :One blood sample from each animal was collected in a clean, dry bottle containing (EDTA) as anticoagulant. 2- Hematological parameters :Red and white cell counts were carried out by using the improved Neubauer hemocytometer. The differential leucocytic counts were also determined The PCV % was determined by using the microhematocrit centrifuge and haematocrit reader.The blood hemoglobin was estimated as cyanomethaemoglobin .G- Serum examination :1- Serum samples :Blood samples were collected by vein puncture from the jugular vein in clean, dry and capped McCartney bottle were left to clot in a slanting position at room temperature. Serum samples were collected after centrifugation at 3000 rpm for 10 min.2- Serum parameters :The activities of Serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase were determined. Serum concentration of urea, Creatinine, calcium, inorganic phosphorus, Sodium and potassium were determined.H- Identification of ruminal protozoa :The identification of rumen protozoa was carried out after their fixation . The characteristic morphology of each genera and species was compared to the description arranged in tables provided by Williams and Coleman for entodiniomorphs and holotrichs.Results The results of the first experiment showed that the cryopreservation trials of sheep ruminal protozoa using the slow interrupted freezing technique were successful with the three cryoprotectants [glycerol, ethylene glycol, and (DMSO)]. However, the viability percentage was significantly different among the three kinds of cryoprotectants. The DMSO in concentration of 5% produced the highest viability. On the other hand, the lowest viability was recorded with glycerol and intermediate viability was attained by ethylene glycol. The difference between DMSO and glycerol was explained by the speed at which they permeate the cell. Glycerol permeates more slowly than DMSO. The use of egg yolk as an additive during cryopreservation did not significantly affect the viability when being used with any of the three cryoprotectants .The results of the second experiment showed a significant increase in rumen ammonia nitrogen concentration at 90, 120 days in control and refaunated groups than defaunated group. This was explained on the basis that the defaunation or reduction in the protozoa population leads to an increase in the bacterial population, which uses ammonia as the source of nitrogen for cell synthesis. Thus more ammonia is being used when the bacterial population is increased. The reduction in ammonia concentration could thus be due to high rate of ammonia assimilation by bacteria, as well as reduced sources of ammonia entering the pool when protozoa are absent or present in small numbers.The total protozoal count showed a highly significant reduction in defaunated animals when compared to control and this significant reduction continued till day 120. The toxic effect of oil drench could be attributed to increasing acidity, resulting from the free fatty acids liberated from the oil and protozoa are more sensitive to pH than bacteria. The refaunation by injection with straw of the rumen protozoa cryopreserved with the DMSO 5% (highest viability) increased the protozoal number progressively until it reached close to control values after 60 days indicating that cryopreservation was a successful procedure to refaunate animals that have been defaunated.The refaunation regained the body weight to almost the control values in all time periods whereas, defaunation significantly reduced the live body weight. These results indicate the importance of rumen protozoa and its effect on digestibility.The ruminal pH in sheep was significantly decreased after defaunation compared with control (faunated) group when measured just before the meal (0 time) and 2 h after the morning meal. This was explained by the major role of protozoa in slowing down the fermentation by ingesting starch grains and taking up soluble sugars and converting them to storage polysaccharides.The hematological parameters including, RBCs count, Hb (g %), PCV% and differential leucocytic count did not significantly change among control, defaunated and refaunated groups. Similarly, serum parameters including urea, creatinine, ALT, AST, AP, Ca, P, Na and K did not significantly change among control, defaunated and refaunated groups.Morphological classification of ruminal protozoa, showed that the most common species that were identified in rumen samples included:1-Diplodinium species (D. dentatum and D. rostatum).2-Isotricha species (I. intestinalis and I. prostoma).3-Entodinium species (E. bursa, E. caudatum and E. simplex).Eudiplodinium neglectum5-Daystrichia ruminantum6-Polyplastrom multivesiculatum.