Factors Affecting Semen Preservation | Animal Husbandry & Veterinary Science Optional for UPSC PDF Download

Introduction

Effective semen preservation is pivotal in various reproductive technologies and animal breeding programs. This intricate process involves meticulous control of factors such as temperature, energy supply, osmotic pressure, buffering, microbial contamination, toxicity, dilution effects, and prevention of cold shock. Understanding and addressing these factors are crucial for maintaining the viability and functionality of spermatozoa during storage, ensuring successful reproductive outcomes.

Factors Influencing Semen Preservation

Temperature Control

• Sperm stored at extremely low temperatures (-190°C) may remain fertile for years, but aging characteristics become apparent.
• Changes in internal and external sperm environments occur during freezing, affecting solute solubility, colloidal structure, viscosity, and motility resistance.

Energy Supply

• Dilution of semen leads to a considerable reduction in fructose, affecting sperm motility and cell maintenance.
• Providing glucose or fructose is essential to meet the energy requirements for sperm function.

Osmotic Pressure Considerations

• Even diluents with equivalent osmotic pressure as plasma may not be isotonic with bull spermatozoa.
• Inadequate isotonicity can lead to morphological and functional damage, emphasizing the need for carefully selected extenders.

Buffering for Protection

• Bicarbonate media saturation with CO2 can cause metabolic inhibition and reduce pH, impacting semen quality.
• Inclusion of catalase prevents the formation of toxic products like H2O2, particularly in the presence of light and oxygen.

Microbial Concerns

• Extending media can create an environment conducive to microbial growth, potentially harmful to spermatozoa.
• Antibiotics are added to counteract the growth of microorganisms in semen extenders.

Toxicity Issues

• Cryoprotective agents like glycerol, while necessary, can be toxic to a certain extent.
• Dead spermatozoa release amino acid oxidase, which, through H2O2 production, can harm surviving cells.

Dilution Effects

• Simple dilution depresses sperm motility, and this can be counteracted by adding amino acids or egg yolk.

Cold Shock Prevention

• Cooling semen rapidly to 5°C can cause cold shock, leading to the leakage of intracellular materials and decreased motility.
• Slow cooling, along with the addition of substances like EDTA, lecithin, and proteins from egg yolk, helps prevent cold shock.

Composition of Diluents

• Maintaining the pH of semen between 6.5 to 7.0 is crucial for storage.
• Phosphate and citrate buffers, such as those derived from KH₂PO₄ and Na₂HPO₄, are commonly used to stabilize pH during semen preservation.

Buffer Solutions for Semen Preservation

Citrate Buffer Composition:

• A 3% solution of Na₃C₆H₅O₇·H₂O or a 4% solution of Na₃C₆H₅O₇·8H₂O, or a 4.7% solution of Na₃HCO₃·11H₂O forms an efficient citrate buffer.
• A 1.3% sodium bicarbonate solution, while effective, is found to be less efficient than citrate buffers for semen preservation.

Selection Criteria for Citrate Buffer:

• Citrate buffer is favored over phosphate buffer due to its ability to disperse fat globules in egg yolk, allowing better microscopic examination of spermatozoa.
• Distilled water for buffer preparation should be double distilled in glass to avoid harmful metallic ions present in water distilled in metallic stills.

Preparation of Complete Dilutor:

• A complete dilutor is created by combining one part fresh egg yolk with four parts of the selected buffer solution.
• Egg yolk acts as a protective agent during cooling, maintains medium viscosity similar to semen plasma, and provides essential nutrients.

Illinois' Variable Temperature (IVT) Dilutor:

• Developed at the University of Illinois, this diluent allows semen preservation at room temperature by temporarily immobilizing spermatozoa with carbon dioxide gas.
• The IVT dilutor includes sodium citrate dihydrate, sodium bicarbonate, glucose, potassium chloride, and antibiotics for preservation.

Cornell University Extender (CUE):

• Foote et al. introduced this dilutor for semen preservation at 23-24°C.
• The CUE composition comprises sodium bicarbonate, sodium citrate, potassium chloride, glucose, glycine, citric acid, and estalose, along with antibiotics for enhanced preservation efficacy.

Semen Preservation at Room Temperature

Citric Acid and Bicarbonate Reaction

• Citric acid reacts with bicarbonate, releasing CO₂, an effective method for preserving semen at room temperature.
• Spermatozoa contribute to CO₂ production via the breakdown of glycine, inhibiting respiration through glyoxylate mechanisms. Catalase acts on hydrogen peroxide produced by spermatozoa from amino acids, allowing preservation for up to 3 days.

Coconut Milk Extender (CME)

• Semen preservation for seven days at room temperature is achieved with Coconut Milk Extender.
• Composition includes coconut milk, sodium citrate dihydrate, nystatin, dihydrostreptomycin sulphate, sulphanilamide, polymyxin B sulphate, mycostatin suspension, sterile catalase, egg yolk, and distilled water.

Preservation at Refrigerator Temperature (4-5°C)

Egg Yolk Phosphate Extender (EYP)

• A dilutor for preserving bull's semen for 72 to 96 hours and buffalo semen for 48 to 72 hours at refrigerator temperature.
• Comprises dilute hydrogen phosphate, potassium dihydrogen phosphate, and distilled water, mixed in equal parts with egg yolk.

Egg Yolk Citrate Extender (EYC)

• A popular dilutor for preserving bull and buffalo semen for up to 72 hours.
• Consists of sodium citrate dihydrate and distilled water, with a mixture of four parts of this buffer and one part egg yolk forming an effective diluent.
• Microscopic examination reveals a clear film of semen with visible spermatozoa.

Egg Yolk Glucose Bicarbonate Extender (RYGB)

• Comprising sodium bicarbonate, glucose, and egg yolk in specific proportions, this dilutor is especially beneficial for buffalo semen.
• Reducing electrolyte content and incorporating metabolizable sugar enhances spermatozoa viability.

Milk Dilutor

• Both homogenized and boiled pasteurized milk serve as effective dilutors, with extension rates up to 1:25.
• Whole or skim milk offers protection against cold shock, attributed to milk protein casein.

Tris-Extender for Prevalent Use

• Tris-hydroxymethylaminomethane (CH₄NO) in combination with glucose or fructose forms an ideal diluter for bull and buffalo semen, surpassing other extenders.
• The composition includes citric acid, fructose, glycerol, and distilled water, with the addition of 20% egg yolk for optimal results.

Preservation at Ultra-Low Temperatures

• Spermatozoa, including cattle, can be preserved at -79°C or -196°C by treating with glycerol, preventing intracellular ice crystal formation.
• Extenders like citrate yolk glycerol, skim milk glycerol, lactose yolk glycerol, and tris-yolk glycerol are used for freezing semen.
• Tris-yolk glycerol is the preferred choice, ensuring better results in the freezing and storing process.

Freezing Techniques

• Freezing is accomplished either in solid carbon dioxide at -79°C or in liquid nitrogen at -196°C.
• Liquid nitrogen has become the standard for freezing and storing semen due to its widespread use and efficacy.

Transportation of Semen

Transport of Liquid Semen:

• The optimal storage temperature for liquid semen is 4°C, which must be maintained during transit.
• Diluted semen is placed in test tubes or small glass bottles, ensuring they are filled completely to prevent air space and the detrimental effects of shaking.
• Proper labeling of each semen tube is essential to avoid identification errors, and corking is done meticulously to prevent water entry.
• Tubes are surrounded by cotton wool or placed inside jacket tubes to avoid direct contact with ice, with sufficient ice in thermos flasks to maintain the required temperature during transit.
• Inseminators commonly carry semen in thermos flasks for short-distance transport, while for longer distances, more advanced shippers with ice and heavy insulation are necessary. Various models, such as the Poona model, Bangalore model, Mathura model, and I.V.R.I. model, have been developed to suit different requirements.

Transport of Frozen Semen:

• Frozen semen is stored and transported in liquid nitrogen at -196°C, allowing for long-distance transport, even across countries.
• Stainless steel containers of different sizes, with double walls and a vacuum space in between, are used to hold liquid nitrogen.
• Canisters inside the container accommodate canes or racks, where ampoules of semen were traditionally placed, but plastic straws are now commonly used.
• The straws are stored in plastic cups of varying sizes and capacities, all immersed in liquid nitrogen in the canisters.
• Regular checks are performed to ensure that at least half the length of the canister remains dipped in liquid nitrogen, replenishing as needed.
• During long-distance transportation, a wooden crate or protective device like hardboard or cardboard may be employed for the liquid nitrogen containers.

Deep Freezing Techniques in Animal Reproduction

• The concept of semen preservation dates back to 1776, where cooling in snow for 30 minutes was observed to render human, stallion, and frog spermatozoa inactive but revivable upon warming.
• Over 200 years, techniques for deep freezing and preservation have evolved, employing reduced temperatures to depress metabolic activity and extend the active life of spermatozoa.
• The development of theories has further refined the understanding of why and how these techniques effectively preserve semen across various species, including cows, buffaloes, sheep, goats, swine, and poultry.

Advancements in Semen Freezing Techniques and Preservation

Understanding the Impact of Low Temperatures

• Low temperatures slow metabolic reactions, extending functionality, but the freezing and thawing process can cause cell damage, prompting extensive research to minimize such damage.

Historical Breakthrough and Basic Diluent Components

• In 1949, British scientists discovered that suspending cells in a medium containing real could prevent the death of spermatozoa during freezing, removing a major barrier to preserving living cells in a frozen state.
• Basic components of semen diluent for freezing include buffer, fructose, glycerol, egg yolk, and antibiotics.

Evolution of Bull Semen Freezing Techniques

• Bull spermatozoa were successfully frozen over 25 years ago, with continuous modifications and improvements in techniques.
• A notable transition from using solid carbon dioxide (dry ice) to liquid nitrogen (-156°C) as a storage environment has been made, providing a more stable condition for preserved cells.
• Early technology related to storage and delivery systems, including the use of glass ampoules, was developed in the U.S.A.

Introduction of Innovations by International Scientists

• French, Danish, and West German scientists introduced the straw technique for semen freezing.
• Japanese scientists developed a technique for freezing semen in pellets, adopted in some regions, allowing the use of smaller inseminate volumes with fewer spermatozoa. This innovation enables more breeding opportunities from a single ejaculate.

Challenges and Slow Progress in Other Species

• While these techniques have been successful in cattle, progress with other species has been slower due to varying responses of spermatozoa to freezing and thawing.
• Different species exhibit variations in diluent requirements, potentially attributed to differences in membrane composition.
• Methodology for Bovine Semen Deep Freezing using Straw Technique:
• The straw technique, utilizing Tris yolk glycerol as the diluent, is commonly employed for freezing bovine semen.
• The diluent preparation involves mixing fresh pH-adjusted Tris buffer with glycerol in specific proportions and maintaining it in a water bath at 30°C for dilution.

Semen Collection, Evaluation, and Dilution

• Semen collections occur in the early morning with pre-collection stimulation to ensure high-quality semen.
• Immediate evaluation assesses volume, color, consistency, and the absence of foreign matter. Samples with over 60% motility proceed to further processing.
• Dilution rates are determined based on motility, sperm concentration, and straw capacity, with a minimum of 2 billion sperm per straw typically fixed before freezing. Diluted semen is gradually cooled to 5°C in a refrigerator.

Semen Processing and Freezing Procedure

 Equilibration of Semen

• Diluted semen undergoes an equilibration period at 5°C for approximately 8 hours.
• This period allows glycerol to exert its beneficial action on spermatozoa, permeating the sperm and modifying ice crystal formation.
• Glycerol enhances sperm resistance to freezing stress by reducing damage from pressure and mechanical effects.

Filling of Straws

• During the equilibration period, an automatic filling and sealing machine or manual methods, often involving a vacuum pump, are used to fill straws with equilibrated semen.
• Polyvinyl alcohol powder is used for sealing French straws, while German straws utilize stainless steel, plastic, or glass heads for sealing.

Cleaning and Drying

• At the end of the equilibration period, straws are carefully rolled and dried using a clean towel to remove excess water.

Freezing of Straws in Liquid Nitrogen Vapor

• Freezing is performed in a horizontally mounted nitrogen container (e.g., LR 320, LR 250) designed specifically for freezing straws.
• Straws are positioned about 4-5 cm from the liquid nitrogen level during freezing.
• The recommended temperatures for freezing in the LR 320 container range from -150°C to -160°C for approximately 7 minutes.
• After freezing, the straws are rapidly plunged into liquid nitrogen for long-term storage.

Post-thaw Motility Examination

• Post-thaw motility assessment of frozen straws occurs 24 hours after freezing.
• Samples with less than 40% motility are discarded, while those with satisfactory motility are transferred to permanent storage containers for preservation.