Next Generation AI - New Developments to Maximize Efficiency Christianne E Glossop, Malmesbury, England AI has been in use worldwide for almost 50 years. As it reaches maturity, the industry is demanding increased efficiency. Where will this come from? One of the fundamental weaknesses of any AI system is that the fixed costs are high, usually amounting to more than 50% of the total cost of a dose of semen. Good house-keeping and careful negotiation with suppliers will reduce variable costs, and this is always a worthwhile exercise; but reducing the fixed costs must be a high priority target for research and development. The only way to have an impact on the fixed costs per semen dose is to produce more doses per boar place effectively to serve more sows per boar. Alongside this objective one must not lose sight of the importance of ensuring consistently good fertility results with AI. The key to enhancing AI efficiency is to find ways of reducing costs whilst never compromising performance. Most commercial studs using 3.0 x 10 9 sperm per dose report an average semen output of 20-22 doses per ejaculate. Collecting each boar approximately 75 times per year, this amounts to a total of 1500 doses per boar per year. Increasing sperm output per unit time, or reducing the number of sperm required per insemination dose both could result in increasing the number of doses produced. The average number of semen doses used per sow per estrus is between 2 and 2.5. A boar s annual semen production of 1500 doses will therefore be sufficient to serve 600-750 estrous sows. Clearly, reducing the number of times a sow needs to be served is also part of the overall efficiency equation. Increasing sperm output per boar Semen quality (expressed as the total number of viable sperm produced per unit time) is influenced by a range of external factors (e.g. environmental conditions, diet, collection frequency), and internal factors (e.g. health, genetics, age) (Glossop, 1995). Efforts to enhance sperm production by manipulating one or more of these factors tend to be carried out on rather an ad hoc basis, with different AI studs operating their own preferred system, the results of which may well not be repeatable elsewhere. Dietary supplements and special boar rations designed to enhance sperm production and boar fertility have also been tried and tested over the years, with variable results (Wilson, 2000). A recent example of such work is the oral administration of the fatty acid Docosahexaenoic Acid (DHA) in combination with the anti-oxidants vitamin E and selenium in the form of Prosperm TM (JSR Clover Ltd). Workers claim that DHA is an essential component of healthy sperm cells, enhancing membrane integrity and tail flexibility, as well as increasing output. In field trials, there was an average improvement of over 100 piglets per 100 sows served with semen from boars on the supplementation regime, representing an 18:1 return on investment. In addition, there was a significant effect on sperm production (Penny et al., 2000). While such observations are interesting and important, and do demonstrate a cost benefit, it is unlikely that this line of research will ever do more than increase sperm production by 10-20%. Reducing sperm numbers per insemination dose Most boar studs are working with an insemination dose of 2.5-3.0 billion sperm, although the early work of Chris Polge (1956) showed us that a minimum of 1.3 billion viable sperm is sufficient. As semen evaluation and preservation techniques are improved there is potential to reduce the number of sperm required per insemination. Improving semen assessment may allow reduction of the insemination dose, as well as promoting standardisation. Reducing the insemination dose to 1 billion cells would have a significant effect on costs, while enabling
superior boars to be spread even further. Techniques in current use rely on estimations of motility, photometric measurement of sperm count and microscopical examination of sperm morphology (Almond et al., 1998). Laser technology is being used to replace both the microscope and the photometer, providing a simple, accurate system ensuring standardisation. Another new technology is computer-aided semen assessment, where image analysis enables the number of moving sperm cells in a sample to be counted, while analysing quality of movement. Other work is concentrating on sperm function tests using differential stains and automated cell counting e.g. the short HOST (hypo osmotic swelling test), which evaluates both the integrity of the plasma membrane in the sperm tail and the status of the sperm head membrane (Vazquez et al., 2000). One major area of new research is focusing on the possibility of reducing the insemination dose by delivering the semen further into the sow s reproductive tract. The extreme version of this concept is the technique of deep intra-uterine insemination. Work from Spain (Gil et al., 2000) has demonstrated that a dose of 20 million sperm may be sufficient if sperm are deposited deep into the uterine horns. While deep intrauterine insemination is a technique which may never be appropriate or acceptable on commercial farms, outside of carefully controlled conditions, and may even have welfare implications in some countries, this demonstrates a significant potential in terms of reducing the semen dose. Other groups in Europe and the USA have also been examining this important and exciting area in a slightly more conservative way. Insemination simply through the cervix, and just into the uterine body can also have an impact on the number of sperm required per dose. There are some concerns about the possible damage caused by the use of extended catheters inseminating deep into the uterus. Studies on the pathology of the uterus, along with a consideration of the effect of such treatment on the long-term breeding potential of a sow are overdue. Enhancing sperm transport through the uterus may have the same effect as depositing the semen deeply into it, with acceptable conception rates resulting from lower sperm dose rate. Recent studies have investigated the role of seminal plasma in sperm transport, identifying oestrogens as a key factor in this process (Waberski et al., 1997). The use of synthetic seminal plasma to enhance sperm transport and fertilization rates is based on the same principals (Lyczynski et al., 2000). Treatment of the sow with PGF2 either by injection or in the semen dose at the time of insemination has been shown also to enhance this process (Pena et al., 1998), as well as possibly having an impact on the timing of ovulation. This area needs further study, particularly with reference to reducing the number of sperm inseminated. It may be of even greater value where the sperm to be used are particularly precious, valuable, scarce (e.g. a particular genotype, or sexed), or in some way damaged (e.g. aged, or frozen/thawed) where motility and viability may be impaired. Intracytoplasmic sperm injection (ICSI), perhaps the ultimate in maximizing the use of a single ejaculate, is really an adjunct to other embryo manipulations. A single sperm is injected through the zona pellucida of an oocyte, thereby effecting fertilization. It is preceded by retrieval of eggs from a donor sow, and is followed by other in vitro manipulations before the fertilized eggs are introduced into the recipient (Cameron, 1998). It offers a complex technique that is extremely efficient in the use of sperm, and would be of particular value where sperm are very precious e.g. from a subfertile or extremely rare sire, or possible sperm which have been sexed. The technique is in use in human infertility cases, but is not yet in widespread use in farm livestock due to the high costs and technical input.
Reducing number of insemination doses per sow served Timing is the single most important factor in successful application of AI. Consideration of the timing of events leading to successful fertilization in the sow demonstrates the importance of ensuring that viable sperm are present within the reproductive tract in advance of ovulation. Ova remain viable in the oviduct for 4-8 hours after ovulation, and exposure to sperm after this time results in reduced fertilization rate and subsequent litter size (Hunter and Dziuk, 1968). One of the most exciting areas of research now in progress is that of real-time ultrasonography of ovarian activity, which is able to pinpoint the timing of ovulation and the factors which influence this important event (Soede et al., 1995, Waberski et al., 1994). Single fixed-time AI should be the long-term goal here, although the commercial AI sector needs to consider exactly how this can be marketed whilst maintaining margin. Reducing wastage It is helpful to consider stud productivity in terms of the number of doses of semen used per boar place per unit time, as this takes into account ordering the correct number of doses and storing them properly on the farm as well as simply maximizing output from the stud itself. While this is of more relevance when a stud is producing semen for in-house use, it is a worthwhile exercise to monitor semen wastage on any breeding unit, regardless of semen source. Semen extenders expand the volume of the ejaculate and preserve its viability for 3-7 days. Understanding of sperm membrane physiology will help to develop longer-life extenders, which will in turn reduce semen wastage. Prolonging the viability of semen for up to 8 days would be of great value as semen could be delivered once a week, reducing transportation costs and wastage (Revell and Glossop, 1989). Another interesting area is the development of low temperature extenders (e.g. 4 0 C) that would simplify semen shipping and storage. An understanding of sperm membrane stability and the physiological requirements of these cells are essential if a longer-life semen is to become a reality. The newer science of micro-encapsulation also is being applied to sperm. This technique seeks to entrap sperm in gel spheres that slowly dissolve to release them, and real progress is now being made in this area (Vigo et al., (2000). Imagine inseminating a sow once at the start of estrus, knowing that populations of live sperm will gradually be released into the uterus throughout the time when ovulation may be occurring! The cattle AI industry relies upon the use of frozen semen, whereas pig AI is based almost entirely upon fresh semen, as fertility achieved with frozen pig semen has been disappointing. Pig sperm are adversely affected by cooling and freezing, and the cryoprotectants used to reduce damage to bull sperm during the cooling and freezing processes do not have the same desirable effects with boar sperm. The most promising techniques available involve freezing the semen in straws or sachets, using egg yolk and glycerol as protectants against damage during the cooling, freezing and thawing processes (Glossop, 1998). The optimum cooling and freezing rates for pig sperm, identification of the best ingredients to protect sperm from damage during the process, and development of the ideal shape for a pig semen straw are all under review. Frozen semen would offer benefits to everyone - simplifying international exchange of genes, and providing all producers with a perfect contingency plan. Current freezing systems are expensive in terms of time and sperm (a frozen insemination dose is double that of fresh semen) - but the benefits are clear. This work may have to wait until the insemination dose has been reduced in order to become commercially viable.
Producing tailor-made end products The ability to predetermine the sex of piglets offers benefits throughout the breeding pyramid. Separation of semen into populations enriched for sperm carrying either the X or Y chromosome is one means of doing this, although embryo-sexing techniques are also being developed. Any technique for sexing sperm must be rapid, and practical in a commercial AI laboratory, while not damaging sperm in any way. Fluorescence Activated Cell Sorting (Johnson et al., 2000) is an accurate though rather slow technique (5 million sperm sorted per hour), and the fluorescent DNA stains used in the process may cause damage to the genetic component of the sperm - a problem that must be taken seriously. Other techniques are attempting to utilize immunological differences between X and Y-bearing sperm to separate out the two different populations en masse, removing the problems of sorting cells individually. Welfare considerations of new breeding technologies As new techniques become available which enhance the productivity and profitability of the swine industry, we must take seriously our responsibilities to the health and welfare of the animals involved. In the UK, the welfare debate has been in progress for 10 years or more. In 1995, the Banner Committee Report raised questions regarding the welfare of practices such as AI and embryo transfer in a farm animals. AI in pigs was included in this review, which in particular highlighted the importance of staff training and veterinary involvement on units practicing such breeding technologies (Glossop, 2000). Stockmanship and animal handling can vary widely and there are, clearly, welfare implications when AI is carried out by badly trained, unsympathetic, disinterested staff. Provided that staff have received appropriate training in the details of reproductive anatomy and physiology, as well as in basic hygiene, these potential problems should be minimized. Excessive numbers of insemination during estrus, dirty technique and late insemination can all contribute to increased incidence of uterine infections and vaginal discharge. It is, of course, easier to AI a sow, which is going off heat, than to persuade her to stand to the boar. As further developments in breeding technologies emerge it is important to consider their welfare implications and make sure that adequate training is given to all those involved in their application. References Almond,G, Britt,J, Flowers,B, Glossop,C, Levis, D, Morrow,M, and See,T (1998). The Swine AI Book, 2nd Edition, Ed. Ruth Cronje, North Carolina State University. Cameron, RDA (1998). Porcine reproduction now and in the future. Proceedings of the 15 th International Pig Veterinary Society Congress,, Birmingham, England, 209. Gil, J, Tortades, JM and Alevia, A (2000). Post cervical insemination. Proceedings of the 16th International Pig Veterinary Society Congress, Melbourne, p399. Glossop, CE (1995). Investigation into boar infertility (2). The Pig Journal, 35, 34-42 Glossop, CE (1998). AI in pigs: production of quality-assured, healthy semen. In Practice, 20, (4), 182-188 Glossop, CE (2000). Animal welfare and the artificial insemination industry. Boar Semen Preservation IV, Beltsville, Maryland, August 2000. Pages 207-211. Hunter, RHF and Dziuk, PJ (1968). Sperm penetration of pig eggs in relation to the timing of ovulation and insemination. Journal of Reproduction and Fertility, 15, 199-208. Johnson, LA, Dobrinsky, JR, Guthrie, HD and Welch, GR (2000). Sex preselection in swine: flow cytometric sorting of x- and y- chromosome bearing sperm to produce offspring. Boar Semen Preservation IV, Beltsville, Maryland, August 2000., 107.
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