SUGGESTIONS TO REDUCE COMMON ERRORS DURING FROZEN SEMEN HANDLING AND ARTIFICIAL INSEMINATION PROCEDURE
India is endowed with huge livestock population reared under diverse production systems and agroclimatic conditions. The country shares 15, 58, 18 and 7% of world’s cattle, buffalo, goat and sheep population, respectively. Increasing pressure for ensuring food and nutritional security to expanding human population on one hand, and shrinking resources (land, water and other inputs) on the other hand, has made it obligatory to obtain more production per unit of land or animal. Traditionally, livestock production in India has been in the hands of smallholders, keeping a small number of different animal species to sustain their livelihoods and to manage their day to day consumption needs. The animals also provide an insurance during economic crisis. However, now the scenario is changing; maintaining low-producing animals in resource constrained situations is no longer economically viable. As such the animal production system is changing towards semi-intensive to intensive mode of production, wherein these get linked to the input supply system as well as marketing channels. Thus, such a transformation in livestock production system has already started and is expected to continue in the years to come. This shift will require superior germ plasm, feed and fodder, and good health care. Since the availability of superior male germplasm, one of the most vital inputs, is limited in the country, the viable option is to use them as optimally as possible so that their superior quality is replicated in large number of females in a shorter period. In order to achieve this objective, the reproduction scientists including the biotechnologists will have to play the major role. Recent developments in reproductive bio-techniques have revolutionized and opened up new avenues for manipulating the reproductive process in livestock for improving their reproductive efficiency. The reproductive biotechnology that has played an unequivocal and very important role in genetic improvement and production enhancement is Artificial Insemination (AI) with cryopreserved semen. The impact of this technology on economic developments of developed nations is very much evident. However, the potential of this technology has not been fully harnessed in several countries, including India. It is an established fact that the technology of artificial insemination using cryo-preserved semen can address major problems being faced by farmers in managing a bull for breeding of their animals. In developed countries, AI is the most common method of intensive breeding in dairy cattle (approximately 80% in Europe and North America), pigs (< 90% in Europe and North America) and turkeys (~ 100%; Thibier and Wagner, 2002). In India, AI is mostly restricted to cattle and buffaloes. Despite having one of the largest networks for livestock breeding, the overall AI coverage in cattle and buffaloes in India is only 29.7 % with an overall meagre conception rate of 35%. The reasons behind the failure to exploit full potential of AI are many. Difficulties in timely delivery of AI, absence of mechanisms to ensure use of semen from certified semen stations, non-adherence to state breeding policy, absence of a mandatory system of animal identification and data retrieval, and poor control over AI technicians are few limitations that need immediate redressal.
The use of artificial insemination (AI) can greatly enhance the production potential of breeding buffalo bulls. However, improper semen handling techniques and equipment maintenance during the breeding process can lead to decreased conception rates and diminish the chances of a productive AI season. Semen quality can be damaged during storage, thawing and handling prior to insemination. Usually, errors made in the handling of frozen-thawed semen are small; however, mistakes in semen handling are frequently addon.
Mentioned below are the areas which often lead to semen handling errors during an AI program.
1. Liquid nitrogen storage tank: Always store the tank away from direct sunlight in a cool, clean, dry, dust-free and well ventilated environment. Accumulation of thick and icy frost in and around the neck of tank, caused by liquid nitrogen evaporation is the first indication of ineffective insulation system of tank resulting in fast evaporation of liquid nitrogen. Monitor the level of liquid nitrogen in the tank with a dip-stick once in a week, and keep a simple log to detect any drastic changes in nitrogen levels. The rule of thumb is that, refill the tank before the liquid nitrogen level falls below 2 inches. The semen is stored in liquid nitrogen at -196oC in the tank. Maintaining correct temperature is important, as a temperature rise and subsequent re-freezing will damage semen.
2. The AI kit: A well maintained insemination kit should always be clean and free of dirt, dust and manure and reduces the chances of introducing infection into a sterile uterus. The kit should contain the following: n Scissors n Thermometer n Thawing tray n Forceps n Singly packed sheaths n AI Gun n Plastic gloves n Clean towel n Thermos / flask with hot water n Apron n 1.5 / 2 liter cryocan with desired semen straws n Tissue paper n Spirit swab in small plastic jar Any deviation from this basic AI equipment will hamper a technician from proper semen handling techniques. Always check to ensure that the AI kit is completely stocked before proceeding with thawing the semen.
3. Proper semen handling (i) Improper retrieval of frozen straws from a liquid nitrogen tank: Always keep the canister below the frost line when locating a straw of semen. Avoid lifting the canister too high or too long during this process. If the straw cannot be located within 5-10 seconds, drop the canister back into the tank and try again. Keep a frequently updated semen inventory with the tank for quick location of bull numbers. (ii) Using bare hands to retrieve straws from liquid nitrogen tank: Use small size forceps rather than fingers to pull semen from goblet. This is primarily for the safety of the technician because liquid nitrogen can cause severe cold burns or frostbite. (iii) Improper thawing temperature and thawing time for frozen semen: Time and temperature are two critical factors in proper thawing of frozen semen. Thaw semen straw in warm water (32-37oC) for 40-45 seconds. Thawing above 40oC may cause it to overheat. Always check the temperature in the thawing tray before pulling a frozen straw from the liquid nitrogen tank. Monitor thaw time with a watch. Never thaw semen in the shirt pocket. Once thawed, straws must not be re-frozen. (iv) Improper drying of straws after removal from warm water: Always wipe the straw completely dry with a clean tissue paper before loading it into the AI gun because water is lethal to semen. (v) Improper cutting of semen straw: Always cut the straw straight and not at an angle at the crimped sealed end. Push the straw into adaptor of the plastic split sheath to prevent semen back flushing into gun during insemination. (vi) Failure to protect semen from direct sunlight: Always protect straw from ultraviolet light with a clean tissue paper to prevent sperm cell damage. (vii) Thawing too many straws at one time or taking too long to inseminate: Thaw only one straw at a time. Inseminate the animal within 15 minutes once a straw is thawed. (viii) Failure to protect AI gun from temperature extremes: Cold or heat shock to semen results in damage to morphology and motility of sperm. (ix) Use of soap or detergent as a lubricant: Soaps and detergents are lethal to semen. Always use an approved non-spermicidal AI lubricant. (x) Failure to pull the plunger back before loading insemination gun: Always pull back the plunger approximately 6 inches before loading a straw into a semen gun. Not doing so will cost a straw of semen. (xi) Failure to identify the target area for deposition of semen: The target for semen deposition is quite small. Accurate gun tip placement is probably the most important skill involved in the whole insemination technique. Inseminators generally identify this target area by feeling for the end of cervix and the tip of the gun as gun emerges through the internal-os. (xii) Improper site of semen deposition: Liquid semen is usually deposited in the midcervix whereas frozen-thawed semen is placed in the body of the uterus. (xiii) Fast or rough deposition of semen: Gently express the semen and not forcefully throw it out. Semen deposition should take 5-7 seconds. (xiv) Improper position of AI gun during semen deposition: While deposition of semen at the suitable place; take care not to draw the gun too far back. (xv) Accidental blocking of uterine horn during semen deposition: During the process of semen deposition take care that fingers of the palpating hand are not inadvertently blocking a uterine horn or misdirecting the flow of semen in some manner. (xvi) Quick and rough withdrawal of AI gun after semen deposition: Pause before withdrawing the gun allowing the semen to get away and then slowly withdraw the gun form the cervix. Rapid gun withdrawal may allow the semen to flow back into the vagina. Therefore, remove the gun slowly from the vagina. (xvii) Vigorous movement of animal during semen deposition: If the animal has moved during semen deposition, stop the semen deposition and correctly reposition the gun tip before continuing with the semen deposition. (xviii) Failure to use a locking-ring on the AI gun: Always lock the plastic sheath into place on the inseminating gun with a locking-ring. Otherwise, the sheath will slip, leading to improper semen placement during insemination. (xix) Sheaths left out in hot weather: Always store plastic sheaths in a cool place out of direct sunlight to avoid irreversible shrinking and curling of plastic sheaths, which renders them unusable. (xx) Taking shortcuts during insemination: It is highly recommended that all technicians attend an annual refresher course before the breeding season begins. Check that all equipment is functioning properly and all supplies are stocked to avoid makeshift improvisations that may undermine a successful AI program.
MAJOR CONSTRAINS AND ISSUES
While most of the gains of using AI technology have occurred in the developed countries, there are considerable opportunities to increase the use efficiency of AI in developing countries including India. Considering the average achievements of artificial insemination in India, as compared to the gains derived in many developed countries, now a time has come to examine the issues hindering its largescale adoption for getting the desired genetic improvement in the livestock population. After a careful assessment, following issues emerged as the important ones to be addressed to realize the most gains out of the AI technology. • Low coverage of AI: At present, animal husbandry departments are the primary providers of AI services (45.7% of the total) followed by Dairy Cooperatives (24.3%) and other agencies (29.9%). Therefore, the present AI delivery system needs to be strengthened and modernized to ensure a targeted coverage of 50% breedable females in the coming years. The major hindering factor is distance of AI services to farmers. The distance to veterinary institutions providing the service either delays or discourages farmers to access quality AI services. The cooperatives are better equipped to reach farmers in time at their door-steps, provide services more efficiently and receive required feedback effectively. The services of other agencies are largely dependent on the funds received from the state governments and other sponsoring agencies. The major issues related to current AI delivery system include difficulties in timely delivery of AI, lack of mechanisms to ensure use of semen from certified semen stations, non-adherence to state breeding policy, absence of a mandatory system of animal identification and data retrieval, and poor control over AI technicians (Gupta et al., 2017). While attempting to answer the query of expanding the AI coverage to 100 percent breedable population, there is an imminent need to examine the economic feasibility and necessity to broaden the AI delivery system to remote areas of the country to cover very low or poor producing nondescript animals. Further, the biggest question before the country is to evolve an effective strategy to reduce the population of low and non-producing animals. Avoiding breeding such animals could be a viable option. Against this view, another option could be to intensify the breeding programme and bring the faster genetic improvement in these animals. However, it is pertinent to mention here that it took almost 3-4 decades to cover only 30% breedable bovine population under AI. Therefore, the possibilities of undertaking such an exercise may need further debate in the face of shrinking resources. • Poor conception rates with AI: In India, the fertility rate from AI is comparatively lower than in other major milk producing countries, where the fertility rates through AI range from 60-72% with an average non-return rate of 60 days (Vishwanath, 2003). Against this, the average conception rate in India still hovers around 35%. Poor conception rate may be one of the major reasons for poor adoption of AI by farmers. Studies have shown that the acceptance level of AI is lower in areas where the farmers complain about poor conception rate through AI relative to natural insemination. Further, detection of estrus (heat) in animals is another challenge to farmers. Each missed estrus in a dairy cow producing an average of 10 liters of milk a day, leads to a direct loss of more than Rs. 8400/- to the farmer in 21 days, before the animal comes in next heat. Each day of an extended calving interval results in huge economic loss (Rs 281 and Rs. 368 in Zebu and crossbred cattle, respectively per day) to the farmers because of not getting milk from these animals but paying the maintenance costs of non-pregnant cow (Abdulla et al., 2017). Inseminating the animals without proper heat confirmation is a major factor for reduced conception rate under field conditions. It has been reported that the percentage of cattle and buffaloes wrongly detected as in oestrus by visual observations were 11.05 and 20.75 per cent, respectively (Kumaresan et al, 2001). About 19% of the inseminations were performed when the plasma level of progesterone was high and the cows were pregnant. Such an insemination of pregnant cows led to an estimated 17% induced embryonic death and/or abortion (Sturman et al., 2000).
The present system of AI delivery, cannot ensure the service at right time due to (i) stationery nature of most AI centers with fixed working hours, (ii) lack of effective communication between farmers and AI service providers, (iii) limited number of AI service providers and (iv) poor training of inseminators. • Limited availability of bulls with high genetic merit and high-fertility: In order to realize the full potential of AI for livestock improvement, it is essential that the semen used for artificial breeding should be from bulls of high genetic merit. The number of breeding bulls required is 8847 as against the present number of 4158 to meet out the current requirement (2020-21) of frozen semen doses for AI (DAHDF Annual Report 2018-19). Another problem lies in the selection of bulls for replacement, as the tool(s) for selection of males at young age with good fertility are yet to be evolved. The semen production ability of a bull is identified after rearing the male calf for 2-2.5 years and subjecting it to semen evaluation. At this point the bulls with inferior semen quality are discarded. Further to know the fertility status of the bull, one has to wait for years together after the production and supply of sufficient doses of frozen semen for field fertility assessment. Based on the conception rate observed after insemination of a large number of females, a bull is categorized as aboveaverage, average or below-average. Keeping a bull for such a long duration in breeding programme and then identifying it as below-average leads to huge costs to the semen stations as well as to farmers. During such evaluation trials, semen from infertile/sub-fertile bulls often delays the conception and consequently cows remain open for several days leading to loss of milk production and productive life. The cost of raising a bull from birth to 18 months was calculated to be approximately US$1188 and the total annual expenses during breeding period were between US$1820 and US$2110 (Valergakis et al., 2007). In brief, a very effective tool(s) and mechanisms(s) for the selection of males at very early age for high genetic merit and high-fertility is needed. • Frozen semen quantity and quality: The National Action Plan aims to increase the milk production to 300 million tonnes by 2023-24. To achieve this target, the requirement of the total number of bovines and frozen semen doses will be very high. Accordingly, both the bull and the semen dose production facility in the country will have to be scaled up to almost double the existing numbers. Although male and female both contribute to the end result of AI, the role of males is far greater because semen from a single male is used to breed several thousands of females. Thus, ensuring the post-thaw quality of semen assumes immense significance in improving the reproductive capabilities of animals. Although there have been several developments in the process of semen analysis, the developments in cryopreservation, extenders and the method of insemination, have not been translated into reality. Poor understanding of the semen biology is one of the limiting factors for successful cryopreservation of spermatozoa. Even today, traditional method of semen analysis is being followed that gives only an idea about few pre-requisite characteristics of spermatozoa to fertilize an oocyte. These tests do not indicate about other important sperm functional requirements. The sperm motility, viability, acrosomal and membrane integrity are mostly assessed to certify the suitability of frozen semen; however the results of such tests do not always correlate with field outcomes (Rodriguez Martinez, 2013). Further, to maintain the frozen semen quality, adequate quantity of liquid nitrogen at required interval is essential, which is also a major problem at gross root level. • Limited or non-availability of frozen semen for non-bovine species: Frozen semen of non-bovine animals (sheep, goats, pigs, mares etc.) is literally not available at large scale, expect in some pockets wherein few institutes produce cryopreserved semen doses and in very small numbers. Although in India, AI in sheep was introduced in late 1940’s and early 1950’s, AI in small ruminants is yet to be undertaken at commercial scale. In small ruminants, either fresh or fresh + diluted + chilled or frozen semen can be used for AI. However, the AI technique must be selected and standardized on the basis of the type of semen planned to ensure its successful use. At present technological backstopping and policy are not available for use of AI in other species including small ruminants and pigs. Few countries even adopt laparoscopic insemination for sheep with good results. Difficulties in semen cryopreservation, estrus detection and insemination procedure are few constraints in the adoption of the AI technology in small ruminants. • Use of Sexed semen for AI: Recently, the demand and desire for use of sexed semen in dairy cattle is increasing. Several Indian states have already started using the sexed semen for AI in dairy cattle. A single dose of sexed semen straw costs Rs. 1500-2000, however, states are making it available at a subsidized rate. Until recently, the sexed semen doses were imported from other countries, but now under Rashtrya Gokul Mission, sexed semen production facilities are being developed at 10 A-grade bovine semen stations. It is targeted to produce 30 lakhs Sexed/Sex Sorted Semen doses annually from these semen stations. The sexed Semen Production has already started at 4 centers i.e. ULDBs Rishikesh, BAIF Pune, ABS Chitale and Mehsana Semen Station. Here, it is pertinent to note that the current average conception rate in dairy cattle when inseminated with 20 million spermatozoa is around 35% in India, which may further go down when 2 million spermatozoa in one sexed semen dose will be used for insemination. So far a total of 9133 AIs has been done in five districts with an overall conception rate of 24.9% under Pilot Project on AI with Sex Sorted Semen. Thus, there is an urgent need to standardize the dose and the site of reproductive tract for insemination with sex sorted spermatozoa in indigenous and crossbred animals. Further, it is also essential to develop the expertise and train the inseminators with low-dose insemination procedures to improve the conception rate with sexed semen. Once sexed semen is available, majority of stakeholders may like to use it for producing females that may again add to decreased availability of quality males. Hence, a stringent policy needs to be framed for using sexed semen, otherwise uncontrolled use of sex sorted semen might skew the sex ratio towards one sex and lead to unforeseen problems in Indian dairy sector.
Therefore, proper use and maintenance of the insemination equipment, proper semen handling, improving the technical skills of the inseminator and keeping accurate herd records are essential for ensuring a successful artificial insemination program.
Compiled & Shared by- Team, LITD (Livestock Institute of Training & Development)
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Reference-On Request.
