CARE,HANDLING & MANAGEMENT TIPS OF FROZEN SEMEN & LN2 CRYOCANE/ CONTAINERS FOR BETTER AI CONCEPTION RATE

Artificial insemination (AI) is a technique in which semen is collected from the male, evaluated, preserved, stored and inseminated to females. The role of man who stands between the cow and bull play an important role. The efficiency of the person handling the semen and depositing in the cow will influence to a great extent on the conception rate in AI programmes. The efficiency of reproduction in dairy cows is one of the most important factors in determining profitability of the herd. To remain profitable, dairy cows must produce one calf at 12 to 14 months interval. In a healthy herd the average number services per conception is 1.6 to 1.7. If satisfactory fertility rate is to be achieved, many events of the reproductive process have to occur in a well balanced manner. It is always possible that certain portion of bovine population either fails to conceive with a single or more number of inseminations, looses the conceptus during gestation or produces a dead calf at delivery. The affected cows may have long calving interval which tend to decrease the average daily production of milk.

Artificial insemination (AI) has contributed enormously to the genetic improvement of the dairy cattle industry in the last 50 years. Frozen semen in 0.5 ml or 0.25 ml straws has become the universally accepted unit of storage and transfer of bovine genetics to cattle producers. While all the care is taken by the semen producers to collect the best quality semen and ship the same in the best possible way, there are times when results are not seen as required. One of the possible reasons is errors in handling of frozen semen.

Factors affecting conception rate——–

For successful AI,fertile semen must be deposited in the healthy cow’s reproductive tract at the right stage of the estrous cycle. Important factors affecting the conception rate include

a) Fertility of the bull selected for semen collection

b) Processing of the semen

c) Storage procedures of semen

d) Estrous detection and insemination technique

e) Time of AI f) Fertility of cow

Heat detection efficiency and inseminator’s skill ——————-

On an average, the duration of estrum in cow is 15 to 20 hours and ovulation occurs approximately 12 hours after the end of estrum. Identification of cows in estrus is very important as it facilitates the breeder to perform AI at appropriate time.

Symptoms of estrus in the cow———–

• Standing to be mounted

• Attempt to mount other cows

• Stringy mucous hanging from vulva

• Increased restlessness

• Drop in milk yield

• Reduced feed intake

• Frequent bellowing

• Frequent urination and tail raising

• Vulval oedema

Estrus detection aid—————-

Advanced but easily available techniques like “fern pattern” of mucous can help to detect correct time of ovulation.

Procedure:

Collect a drop of cervical mucus, place it on a glass slide and spread it evenly. Dry it in air and examine under microscope (10X) for different fern patterns.

Three types of fern patters are,

Typical- clear fern leaf like appearance. The branches are well marked and have bright and thick boundaries.

Atypical- mixed type appearance. Fern branches remain discontinuous and are not well cut out into further branching.

Nil type- no fern like appearance.

Interpretation:———–

Early heat: fern patterns are scattered and are small in size. Branching is thin and fine.

Mid heat: arborization or crystallization is visible through out smear. The branches are well marked and have bright and thick boundaries. Tertiary or quaternary or quinquinial branching is observed. This type of pattern is generally referred as typical.

Late heat: Crystallization is not typical. Fern branches are discontinuous and not well cut into further branching. There is an increased infiltration of lymphocytes in the smear also

Time of AI and conception rate in cows ——–

The influence of time of insemination on conception rate in cows is furnished in the following table.

Time of AI —Conception rate

Start of estrus– 44 %

Middle of estrus—- 82.5 %

End of estrus —75 %

12 hrs after estrus— 32 %

24 hrs after estrus— 12 %

36 hrs after estrus—- 8 %

48 hrs after estrus—– 0 %

Post- partum breeding—————-

After parturition, adequate sexual rest is a must for cows to the uterine involution. The average time required to complete uterine involution in cow is 45 to 60 days. Therefore, cows should be bred after 60 days postpartum.

General management of females ———

Nutritional factors such as energy, protein, vitamins and minerals needed for successful reproduction are the same as those needed for maintenance, growth and lactation. Deficiency or excess of any of these components which is serious enough to affect reproduction as well as other physiological functions.

Functional disorders in females ———

• Anoestrus- may be physiological due to the effect of season, nutrition, lactation stress, ageing, etc. or may be pathological due to inflammatory conditions of ovary.

• Atypical estrus like short estrus, prolonged estrus, split estrus, nymphomania and silent estrus etc.

• Ovulatory failure

Role of inseminator——————

The present conception rate for AI with frozen semen at the field level isreported to be low. The main reason isimproper handling and / or deposition of the semen. Thawing and insemination are the two important phases to be handled by the inseminator with utmost care. With chilled semen, deposition of semen either in cervix or uterus results in same conception. While with frozen semen, if semen is deposited in the vagina conception is very low, if deposited in cervix the conception rate is lowered and optimum conception occurs with deposition in the body of uterus. The service ofskilled inseminator is essential in maintaining optimum fertility in the field. It has been observed that inseminators who had been inseminating for many years were only 25 % accurate in semen deposition. Though it is very difficult to motivate experienced inseminators it certainly demonstrates the need for retraining.

Thawing of semen —-

Theoretically the faster a sperm is frozen; the more rapidly it should be thawed for optimal survival. To bypass crystallization zone, thawing must be rapid and uniform. The most convenient method is to plunge the straw in warm water. The temperature should be 38- 40 °C for 60 seconds for optimal survival of spermatozoa. During thawing entire straw must be completely submerged in water bath.

Procedure for loading the AI Gun——————-

• Identify the canister from which the desired semen is to be taken. Ascertain the colour of the straw by reading identification tag.

• Remove the lid from the container and lift the proper canister up to the level of the frost line. Never lift the canister above the neck level.

• With a help of forceps, grasp an individual straw and remove it, at the same time lower the canister immediately back into the container. If you are unable to take the straw within 10 seconds, lower the canister back to nitrogen, wait for some time and make next attempt.

• With the wrist movement give one or two jerks to the straw to expel liquid nitrogen trapped at the end of factory seal.

• Dip the straw into a clean water bath at 37- 40 °C for 60 seconds.

• Remove the straw from bath and dry the straw with a clean tissue paper or cotton. Inspect the straw carefully and discard straw with cracks or defective seals. Semen must never come in contact with water. • Place the straw in the chamber of insemination gun. To obtain a perfect fit, it is essential that the laboratory seal be removed by cutting at right angle through middle of the airspace. Make sure that the clipped end of straw has a straight clean cut with no jagged edges. Straws cut at other angles will result in back flow and wastage of semen at the time of insemination.

• The post thaw survival of spermatozoa is poor. For maximum reproductive efficiency, thawed semen should be used immediately. Therefore, do not thaw more than one straw at a time.

Precautions while inseminating a cow—————

• Work under very hygienic conditions, handle semen and AI equipments correctly and do not try your own Lykascope Typical fern pattern at estrum methods.

• Introduce the AI gun first at 45 ° angle, then horizontally and glide along the dorsal side of the vagina. Never use force while introducing the AI gun. This would lead to injuries in the reproductive tract and affect the conception rate. If an animal jumps or falls down, withdraw the AI gun immediately to avoid injury to the cow.

• Insemination of a cow should not be done in hot climate, as pregnancy can never be established.

• Remember that poor fertility with frozen semen is due to improper handling or improper deposition of semen by the inseminator

Efficiency of inseminator————–

Artificial insemination is an unnatural act to which cows may react unfavourably. Extreme excitement or irritation at this time may upset the delicate nervous and hormonal relationship. Practical experience shows that careful, skillful technique is a must for successful AI programme. Some trained inseminators have lower than normal conception rates even though they are able to deposit the semen at the right place. These persons usually are too rough in their technique. For the best psychological cooperation from the cow irritation and excitementshould be minimized, both before and after manipulations of the genital system

Safe handling of liquid nitrogen (LN2 ) —————–

• LN2 should be handled carefully as it can produce “frost bite” on the skin similar to burn, even if it is in contact for more than few seconds.

• Cold vapour of LN2 can damage delicate tissues such as eyes if exposed for a short period. One should not look into an open LN2 container without eye protection.

• Boiling and splashing always occur when filling up a warm container. Always perform these slowly to minimize boiling and splashing and keep your eyes away from the container.

• Always use stainless steel tongs with long handles to remove any object immersed in LN2 . loose fitting gloves made of woolen or leather can be used for handling. Cold metal may stick to skin and tear flesh when attempts are made to withdraw the cold objects from bare hands or fingers. Specially made cryo-gloves are also available for this purpose. Care should be taken to avoid spilling of LN2 into shoes.

• Use containers specially designed to hold LN2. All containers have vent orsafety device to allowthe escape of nitrogen vapour. Inadequate venting or closing tightly can result in excessive gas pressure, which can damage or burst a container. Use only the supplied by the firm; never plug the containers tightly

Ventilation —————

Always handle liquid nitrogen containers in a well ventilated area to prevent excessive concentration of gas. Excessive amounts of nitrogen reduce the concentration of oxygen in the air. When the oxygen level goes below 20.5 % can cause asphyxiation. A person can become unconscious, without sensing any warning symptoms such as dizziness.

Transferring of liquid nitrogen—————

Use plastic funnel while pouring liquid nitrogen into another smaller container. The top of the funnel should be partly covered to reduce splashing. When it is not safe to tilt a container, use a discharge tube to remove liquid nitrogen.

Care and maintenance of containers ——————

The cryogenic containers are double walled vessels, with annular space evacuated and sealed. In addition several types of insulation viz., vacuum alone, expanded foam, gas filled powder and fibrous material are used as thermal insulators. The outer walls of containers are made of stainless steel, carbon steel or alluminium alloys.

• Extreme care should be exercised in handling containers.

• The cryogenic container is specific and they are meant to store only the particular liquid for which they are made.

• Welding or piercing the container wall is dangerous.

• Keep the container always in upright position.

• Protect container against shock and rough handling. During transport support the container with soft padding (rubber, thermocole etc.)

• Protect against direct sunlight and hot blowing winds.

• Filling of warm container should be slow.

• Avoid frequent cooling and warming of containers. Thermal stress may cause so much strain within it that the inner wall of container may crack. Appearance of moisture on the outer wall of container is a sign of damaged container. Do not dry the container meant for regular use.

• Find out the evaporation rate for each container, so that the periodicity of topping could be organized. • When the vacuum disappears, the insulating capacity is lost. Repairing of damaged containers has to be done by the manufacturer only. Hence, containers should be handled very gently particularly during transport in vehicles.

Assessment of liquid nitrogen level——————–

The evaporation rate of liquid nitrogen varies between containers (even among the same make and capacity), temperature of the room in which it is stored and number of times it is opened. Hence, periodic checking of the level of the liquid nitrogen is essential. The minimum level of liquid nitrogen should keep the straws completely submerged in liquid nitrogen. To measure the level, take a dipstick (slender stick made of wood) and gently lower it into the container and rest it at bottom. Hollow tube should not be used. After 5 – 10 seconds, take it out and wave in the air. The atmospheric air condenses as a frost on the stick to the level of liquid nitrogen. Read the level 1 cm below the end of frost.

Proper semen handling procedures from locating the semen in the liquid nitrogen tank to entering the cow’s reproductive tract need review periodically.

Everyone should establish a routine for handling frozen and thawed semen that does not injure sperm cells and lower conception rates. Usually, errors made in the handling of frozen – thawed semen and the equipment used for artificial insemination are small. But the mistakes in semen handling frequently add up meaning their effects on semen quality will be magnified many fold.

Few things must be taken into consideration as farmers/inseminators get ready to thaw the semen and inseminate the cows/heifers.

Coordinate rapid transfer of semen between tanks. Involve two people and arrange tanks side by side. If possible fill the tanks with nitrogen before transfer. Raise canisters only to a level necessary to locate the rack of semen to be transferred.

Develop a semen inventory system and mount it on the wall above the tank.

Try to keep semen from one bull on each rack. Such systems help avoid unnecessary searching and exposure of semen to dangerously high temperatures within the neck region.

When preparing to thaw semen raise the canister into the lower portion of the neck where the specific rack of semen can be grasped. Lower the canister further into the neck. Secure the rack as low as possible in the neck, thus protecting the other straws from thermal damage. If straws cannot be easily removed from the plastic goblet, bend the top tab of the rack to a 45° angle. This reduces the chance of bending the straw.

Use tweezers to transfer the straw to the thaw bath. Quickly lower the rack of semen and canister into the body of the tank.

Thawing of the semen within the straw starts to take place at room temperature after only four seconds, and as a “rapid thaw” is critical to obtaining minimum damage to the sperm during the thawing process, damage will occur if the thaw takes place at room temperature.

Keep insemination equipment clean, dry and ready at all times. The necessary equipment includes Thaw Unit,Thermometer, Lube, Gloves, Insemination gun, Paper towels, Cito cutter or scissors, Sheaths and Tweezers

The Thaw unit is one of the main equipments required to get the semen ready for AI. Either an electric thaw unit or a good reliable thermos is necessary to thaw semen.The goal is to maintain constant water temperature.

Occasionally check the accuracy of the thermometer.

Transfer straw immediately to thaw unit. The temperature of water should be 95-98 degrees F or 35-37 degrees C. Thaw straw for at least 40 seconds but not for more than 15 minutes.

Do not attempt to thaw semen at temperatures greater than 98 degrees F (37 degrees C). Thaw units of semen individually or max 3 straws as thawing more straws does compromise fertility.

With tweezers, remove the straw from thaw unit and dry the straw, using a clean paper towel. Place the straw in the fold of the towel to dry. A small drop of water can be lethal to sperm.

Shake the air bubble from the middle of the straw to the crimped end. Cut off the crimped end of the straw with a scissors or Cito cutter. Cut the tip of the straw squarely, through the air space below the crimp to achieve a 90 degrees cut. An angle cut may prevent the straw from fitting securely into the sheath. Check to see that the straw is firmly seated into the plastic adaptor or tip of the sheath depending on the type of inseminating device/AI gun that is used.

When assembly of the insemination rod is complete, gently depress the syringe to remove the air space at the upper end of the straw. Eliminate the chance of cold shock by warming the inseminating rod and sheath to body temperature (do not use water for this purpose). Use a paper towel and warm by use of friction. Must be done prior to the start of thaw process.

1. Handle the thawed semen and prepare the insemination rod in a warm environment. Wrap the assembled insemination rod in a clean, dry paper towel and tuck it within your clothing for transport to the cow. Inseminate the cow within a few minutes after the semen has been thawed. Never take shortcuts when handling semen or inseminating a cow. Pay attention to detail. Never experiment on your own.

Five most common errors people commit when handling frozen semen and getting ready to inseminate the cow.

1. Raising the canister containing the semen canes above the frost line of the tank (frost line is usually 4 to 5 inches from the top) and removing the semen from the cane using fingers not tweezers. Exposing frozen semen to elevated temperatures in the neck tube of the tank has the potential to cause sperm damage.

2. Improper thaw bath temperature. Either not using a thermometer to obtain thaw bath temperature of 95 deg F/37 deg C or using a thermometer that needs adjustment, thus not obtaining the desired water bath temperature.

3. Not timing thawing. Frozen semen should be in 95 deg F water bath for a minimum of 40 seconds for proper thawing.

4. Straw not dried completely prior to placement in the insemination rod.

5. Straw not cut at proper distance from crimp sealed end (middle of air bubble) at a right angle straight across the straw to prevent semen feedback inside the sheath and insemination rod.

If semen is not thawed properly to begin with, the technician doesn’t need to worry about cold shock as “Dead sperms do not get cold shock”..!

Other considerations to get the best results———-

1. Handle animals gently to avoid unnecessary excitement before, during, and after breeding. Undue excitement may adversely affect sperm transport within the female reproductive system causing a lower conception rate.

2. Breed animals based on standing heat, remembering to breed the animals 10 to 12 hours after the beginning of standing behavior.

Although semen costs are only a small fraction of overall dairy farm expenses, the absolute value of the semen inventory at any given time may represent a significant sum of money. Semen is half the investment in a dairy farm, without which the next generation will not be born and hence no milk. It is important to take good care of the semen as well as the tank in which it is stored.

Liquid nitrogen is an atmospheric gas in liquidized form. It is a cryogenic fluid that can cause almost instant freezing when it comes in contact with living tissue. A liquid nitrogen tank keeps its materials well below the freezing point of water. Contact with skin would cause instant frostbite.

Semen is stored in Liquid Nitrogen Tanks (LN2) and the temperature is -195 deg C at the bottom of the tank and there is a gradient from top to bottom.

Although tanks are designed to be sturdy and dependable, they are more fragile than they appear. They require regular care and attention to ensure they function properly for many years. Failure to do so can lead to lost inventory, or reduced pregnancy rates because semen quality is compromised.

Few tips to take care of the LN2 Tank ——

1. Store the semen tank in a dry, well-lighted and well-ventilated area but out of direct sunlight. Tank must not be put in a place where there is excessive water or chemical use. Observe the tank daily. Once a tank “goes bad” the nitrogen is lost very rapidly. Develop a plan to have an alternative semen tank available in case your tank is damaged.

2. Keep the tank elevated above the concrete floor or other wet and poorly ventilated surfaces. Corrosion of the outer shell shortens the functional life of the tank and possibly causes tank failure.

3. Avoid excessive movement or abuse of the tank. Any stress cracks (which normally appear on the neck), must be taken seriously.

4. Frost is one of the key external warning signs prior to tank failure. A properly functioning tank will not develop frost on the outside. Frost buildup around the outside top of your tank, particularly around the neck, indicates a vacuum loss – which results in rapid volatilization of the liquid nitrogen. Semen quality can be compromised under these circumstances. Keep in mind that this frosting usually happens quickly and doesn’t last too long because the liquid nitrogen has escaped.

5. Routinely monitor nitrogen levels and keep a record of nitrogen usage.

Remember even new tanks can have defects and fail. The temperature in the neck of the tank becomes warmer as the liquid nitrogen level in the tank decreases. Never let the tank go dry.

6. The tank’s cork is handled frequently every time semen is retrieved, it should be inspected monthly to make sure it is doing its job. A poor quality cork increases the boil-off rate by 20 percent to 30 percent. That’s because insulation properties decrease for these corks and more heat gets into your tank. Exposing an open tank to the environment and wind can multiply the boil-off rate by up to 8 times the normal amount.

One person should be made responsible for the tank, create a checklist of things to do and check along with a timetable. This will help in plugging any loopholes and minimize damage to the investment.

Retrieval techniques also help in limiting the damage to the semen

When removing a straw from a liquid nitrogen tank, it is imperative that the technician keep the canister, cane and unused semen straws as low as possible in the neck of the tank. A best management practice is to keep all unused straws below the frost-line in the neck of the tank.

Keep in mind that although the temperature of liquid nitrogen is -195 degrees C, there is a temperature gradient in the neck of the tank. For example, a tank with a neck tube that measures six inches long may have a temperature of -75 degrees C in the middle of the neck (3 inches below the top), while the temperature at 1 inch below the top may be + 2.2 degrees C. Reports have shown that sperm injury (as judged by sperm motility) occurs at temperatures as low as -78.8 degrees C. Furthermore, injury to sperm cannot be corrected by returning semen to the liquid nitrogen.

Use tweezers/angled forceps to retrieve the straws and keep the canisters as low as possible and do not use the fingers as they might get damaged due to frostbite. If the technician is unable to retrieve the straw in 10 seconds, the canister should be lowered and allowed to re-cool for 20 seconds before trying again. More time in a warm zone leads to partial thawing and can compromises the semen quality.

Reference-On request