310 Extender

310 Extender - TECH 102: ’ EXTENDER PRINCIPLES AND...

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Unformatted text preview: TECH 102: ’ EXTENDER PRINCIPLES AND PREPARATION Accompanied Reading: Juan C. Samper E uine Breedin Mana ement and Artificial Insemination CHAPTER 2: Sperm Physiology “The Equine Sperm Cell”, pages 30 — 33 “Application of Sperm Cell Biology to Equine Breeding Management”, pages 36 — 37 CHAPTER 9: Artificial Insemination “Processing and Preserving Fresh Semen". pages 1 18 — 120 PRINCIPLES OF EQUINE REPRODUCTION Sperm cells are perhaps the most unique cell structures produced by mammals. They are independently propelled, possess a haploid number of chromosomes, highly specialized in function. and have limited capacity for repair. Because of their specialized functions and extra ordinary cellular characteristics, these cells are perhaps some of the most unique in the biological system. In addition, a noteworthy distinction between sperm cells and other biological cells is their need for two different environments to complete maturation. While sperm, for the most part, become fimctional during spermatogenesis and storage in the epididymis of the stallion, these cells also require proteins and enzymes present in the mare’s reproductive tract to become fully capable of oocyte fertilization. According to Amann and Graham, spermatozoa must develop and retain five characteristics in order for them to successfully fertilize an oocyte.4 They must have: 1. Metabolism 2. Progressive motility 3. Enzymes 4. Proper distribution of lipids in the plasma and acrosomal membranes 5. Proteins in the plasma membrane critical for the survival of the spermatozoa in the female reproductive tract. In vitro and in vivo environmental physiological effects imposed on sperm cells after ejaculation critically determine whether these cells will survive and successfully fertilize an ova. Thus, it is necessary to acquire a brief understanding of the biological and physiological principals affecting sperm survivability while being stored fresh for artificial insemination or processed for cooling. The principal damaging agents affecting sperm viability during processing are: temperature, oxidation, toxins. virulents, and starvation. Temperature The most critical of the handling and processing principals is the need to maintain a physiological temperature in which the spermatozoa can survive, but more importantly maintain their capacitation and fertilization capabilities. However, regardless of the detail of collection. a limited amount of cooling occurs. Thus. the responsibility of the collector is to collect and place the collection into an appropriate thermal environment that will maintain metabolism and cellular structure. Rapid changes in temperature induce irreversible anatomical and physiological damage. Thermal effects on spemiatozoa are listed in Table l. 10 NOTES llllllllllllllllllll TECH 102: EXTENDER PRINCIPLES AND PREPARATION NOTES Characteristic 22°C 27°C 32°C 37°C Motile Sperm (%) 49 63 64 67 Progressively Motile Sperm (%) 33 46 53 58 Table 2: Thermal Effects on Motion Characteristics of Spermatozoa4 For example, a handler places a freshly collected semen sample onto a cold counter. The effect of rapid cooling, considered a rate 2 0.050C/min, will result in irreversible damage affecting spemtatozoal cellular structures and function. Damage caused by thermal variation includes abnormal swim patterns, rapid loss of motility, acrosomal and plasma membrane damage, reduced metabolism, and loss of intracellular components.4 Appropriately, this damage is referred to as cold shock. Oxygen Deprivation and T oxicigy It is commonly thought that spermatozoa must utilize a direct source of oxygen for survival (aerobic respiration). While equine spermatozoa require aerobic respiration they are capable of utilizing both aerobic and anaerobic respiration processes. While normal atmospheric oxygen does not present a direct threat to fertility rates of these cells with same day insemination, new studies available indicate that extended exposure to oxygen may actually be harmful, due to lipid peroxidation. Magistrini et a]. has suggested that oscillating tubes at 5 turns/min increasing the distance between sperm cells and the oxygen environment in a 2— mL tube, on a continual basis, may actually increase motility. The experiment concluded that continual circulation of spermatozoa into and away from direct 02 contact actually improves motility.8 However, one must be careful as not to restrict normal atmospheric 0; infusion by sealing the container entirely, since equine sperm cells rely heavily on aerobic respiration. Restricting oxygen availability in the collection vessel eventually leads to oxygen deprivation, forcing cells to turn to anaerobic respiration processes, in turn increasing the levels of lactic acid production resulting in fatal seminal plasma pl—I changes. While extended exposure to oxygen may be toxic. this research has not yet been thoroughly investigated, and should not be of concern in artificial insemination programs where mares are inseminated at the fami. Rather. this should be considered when utilizing cooled semen practices. but remembering that this may not be practical outside research development. l l _. PRINCIPLES or EQUINE REPRODUCTION Cellular Waste Products As with all organisms that utilize metabolizable energy sources (i.e. fructose, glucose, sucrose, pyruvate), extracellular by—products are produced that may directly/indirectly affect environmental conditions. Glucose is the main source of metabolizable energy for the equine sperm cell. Thus, the main by-product from the glycolytic cycle is lactic acid. This acid accompanied by peroxides produced from lipid oxidation, affect seminal plasma pH as well as motility and viability, plasma membrane integrity, structural integrity of organelles, and metabolism. Virulence: A stallion’s penis maintains a natural microflora of bacteria. If growth of this microflora is disrupted virulent pathogens may begin to develop that infect the endometrium during copulation, commonly referred to as endometritis.l4 The three main groups of classifiable diseases are: 0 Viruses 0 Bacteria 0 and Protozoan However, bacteriologicals are of most concern in semen collection and extender preparations. The most common bacteria causing endometritis are Klebsiella pneumoniae, E. coli, Streptococci, Pseudomonas aeruginosa, Staphylococcus 51)., and Heomophilus equigenitalis.2 Effect on motility after storage at «5°C for 12h Antibiotic Arnikacin Sulfate Gentamicin Sulfate Streptomycin Sulfate Potassium Pen 0 Sodium Pen G Ticarcillin Na; Polymixin B Sulfate Table 3: Effects of Various antibiotics added to milk-based extender on sperm motility after storage.N NOTES TECH 102: EXTENDER PRINCIPLES AND PREPARATION Starvation NOTES It has been known for quite sometime that the glycolytic breakdown of glucose is the principle energy source for metabolism in equine spermatozoa. However, the sperm cell must utilize direct sources of exogenous sugars. as they are unable to metabolize glycogen stores. Endogenous compounds used in metabolism represent approximately 10% of the ATP production of the cell.4 Thus, 90% of all metabolizable energy sources come from extracelluar sources, such as glucose, fructose as well as other substrates like lactic acid. glycerol, fatty acids, and amino acids. These sugars are mediated across the plasma membrane via specific transport proteins for use by the mitochondrial cells in the midpiece of the cell. Therefore, a principle concern in the preservation of semen, whether fresh, cooled, or frozen, is a consistent supply of usable energy sources. EXTENDER EFFECTS AND ADVANTAGES Seminal plasma is an essential transport medium during natural mating; however, plasma itself is a poor substitute for artificial extenders. Furthermore, as with the mare, stallions should be treated as individuals, and selection of the extender to be used with individual stallions should be based on survivability and progressive motility. Thus, one should always keep in mind that stallions respond differently to the various types of extenders. When one is evaluating semen for storage or insemination with an appropriately diluted ejaculate, analysis should be based on the quality of the raw ejaculate, dilution rate, rate of cooling, storage temperature, storage time, container, and type of extender. The overlying principle of extenders is to artificially prolong the life of spermatozoa by stabilizing enzymes, protecting sperm cells from unfavorable environmental conditions, etc; thus, dilution of seminal plasma with an appropriate extender is essential for maintaining spermatozoa for prolonged periods of time. Extending raw semen with an appropriate extender will: '6 1. Permit effective antibiotic treatment of semen. Helping to prevent the transmission of potentially pathogenic organisms. 2. Extend the life of spermatozoa by providing a more favorable environment. 9.) Protect spermatozoa from unfavorable environmental conditions. 4. Increases the volume of inseminates. 5. Enable prOper evaluation of spemi motility. 6. Provide nutrients to attend the life of spermatozoa. DJ PRINCIPLES OF EQUINE REPRODUCTION An effective extender should minimize harmful environmental conditions, such as cold shock, toxic by-products. infective agents, and inadequate energy sources. A good extender will provide:16 1. Compatible osmotic pressure. (300 - 400 mOsm) 2. Proper balance of mineral elements. (electrolytes and non-electrolytes) Proper combination of nutrients. (glucose, fructose, pyruvate, etc.) DJ 4. Neutralization of toxic products. (lactic acid) 5. Protection against temperature. 6. Stabilization of enzyme systems and integrity of membranes. However, advantages of extenders are not solely limited to semen preservation. Uses of extenders provide the breeder with a means of providing quality semen that is disease and microbe free. Due to the added antibiotic agents, mares problematic with endometritis can be more effectively managed and bred.8 Likewise, it has been shown that extended volumes and the resulting concentration reduction of spermatozoa reduces inflammation after insemination. 14 NOTES TECH 102: EXTENDER PRINCIPLES AND PREPARATION TV es 0 Extenders NOTES Extenders vary widely based on their composition and function. There are extenders for use with fresh semen at the farm. basically a buffered solution with a glucose additive and an optional antibiotic. Then there are extenders for cooling and freezing of spermatozoa. These extenders generally have a glucose additive. buffering components, and glycerol. For the most part, simple skim milk extenders are the most widely used with fresh semen analysis and insemination at the farm. The advantage of the skim milk extender is that it can be prepared at home on a regular stove and skim milk, preferably without vitamin additives, purchased at a local supermarket. This extender type is the focus of this laboratory. Other extender types include: 0 Cream-Gel Extender - Skim Milk-Gel Extender 0 Skim Milk glucose + sucrose-bovine serum albumin Extender 0 Skim Milk + egg yolk o Citrate-EDTA and Glucose—EDTA Centrifugation Mediums - Freezing Extenders: F R5 Extender and Colorado (CO) Extender INSTRUCTIONS FOR PREPARING A BASIC SKIM MILK EXTENDER + OPTIONAL ANTIBIOTIC 1. Prepare a hot water bath using a lOOO-mL beaker or large pot filling them with deionized water to prevent scorching of the milk. 2. Measure lOO-mL of Skim Milk of choice (this can be liquid milk from the store or dried skim milk powder. preferably nonfortified) and place into a Erlenmeyer flask or other glassware that can be partially submerged into the hot water bath. DJ Place a thermometer into the Erlenmeyer to measure the temperature. 4. Begin heating the water, using a Bunsen burner or other heating device, and CAREFULLY monitoring the thermometer. o The temperature needs to be kept between 92-950C. Temperatures greater than 950C will denature proteins. 0 DO NOT ALLOW TO BOIL. 5. Heat milk at the temperature specified in Step 4 above for 10 minutes, PRINCIPLES OF EQUINE REPRODUCTION 10. 11. 16 0 DO NOT start your time until the milk has reached 92°C. 0 Allow milk to heat for a full 10 minutes at or between 92—9500 After heating for 10 minutes at the specified temperature, allow to cool to room temperature. Using vacuum filtration or other filtration device, filter any clumps that occurred during heating. If adding antibiotic of choice, warm 10-15 mL of Deionized Water on a hot plate and add antibiotic powder into the water by infusing liquid antibiotics or breaking the capsule. Warm water until the antibiotic has completely dissolved. Add to the filtered Skim Milk and incorporate thoroughly. Pour into desired storage containers and freeze until use. NOTES TECH 102: EXTENDER PRINCIPLES AND PREPARATION ln redients Quantitv Sanalac (instant nonfat dry milk) Glucose. monohydrate Sodium bicarbonate (7.5% solution) Gentamicin sulfate (reagent grade) Distilled Water Table 4: Composition of Nonfat Dried Milk Solids-Glucose Extender.‘ Extender Compostion of CO and FRS Freezing Extenders Ingredients Quantitv Sterile skimmilk Solution of salts and sugars Glucose Lactose Raffinose Sodium citrate dihydrate Potassium citrate Deionized water to make 50—mL Egg yolk Glycerol Ticarcillin Table 5: Composition of FRS Freezing Extender.16 Ingredients Quantity Dried Skimmilk 5.15 g/ 100 mL Solution of salts and sugars NaCl 18.5 mM KCl 5.0 mM KH1P04 0.6 mM NaHCO; 17.8 mM MgSO4 1.2 mM Hepes 5.0 mM CaCll ' H30 mM Fructose 42 mM Glucose 18.5 mM Na Pymvate 0.6 mM Na Lactate 6.55 mM BSA 1.6 mg / m1. Egg yolk 2% Glycerol 3% (vzv) Table 6: Composition ofCO Freezing Extender.” l7 PRINCIPLES OF EQUlNE REPRODUCTION COMMERCIAL AVAILABILITY NOTES All extenders are generally commercially available for purchase. The most popular extender of choice is the E-Z MixinTM Extenders available from Animal Reproduction Systems. Other suppliers include: Colorado State University and Exodus. E-Z MixinTM choices include: 0 13-2 MixinTM CST — contains Arnikacin 0 13-2 l‘viixinTM OF — contains Polymyxin B Sulfate o E-Z MixinTM BF — contains no antibiotics or addition of antibiotic of choice 0 E-Z MixinTM CST+ ~— contains Potassium Penicillin G in a separate capsule + Arnikacin 0 13-2 MixinTM OF+ — contains Potassium Penicillin G in a separate capsule + Polymyxin B Sulfate 0 15-2 MixinTM BF+ — basic formula + Potassium Penicillin G in a separate capsule c E-Z MixinTM BFT —- contains a separate Ticarcillin capsule Suppliers Animal Reproduction Systems 14395 Ramona Ave. Chino, CA 91710 (800) 300-5143 m~warssalescom Exodus 5470 Mt. Pisgah Rd. York. PA 17406-9200 (877) 396-3874 mm:exodusbreederscom ...
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310 Extender - TECH 102: ’ EXTENDER PRINCIPLES AND...

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