A World Survey of Artificial Breeding II. Technique

Br. vet. J. (1969), 125, 580

A WORLD SURVEY OF ARTIFICIAL BREEDING II. By

TECHNIQUE T.

BONADONNA

Instituto Zootecnia Generale, Milan, Italy

TECHNOLOGI CA L PROBLEMS AND IMPROVEMENTS

During the past 5 years the applied technology of A.I. has been passing through an interesting phase of increased specialization, with the more comprehensive adoption of the results of investigations in the whole field of animal reproduction-in biology, genetics, biochemistry, biophysics, anatomy and now also in mechanics and electronics.

(a) Semen collection Techniques of collecting semen have changed little over the years. The artificial vagina is still the instrument most used, and is always preferable since it is a method that we have defined as "paraphysiological"; that is, it allows normal male libido to be maintained, and also the integrity of the external reproductive apparatus as well as the biological characteristics of the semen. Electro-ejaculation is an interesting and suitable method, but an exceptional one, since it should be used only when the artificial vagina cannot be employed. It is generally held to be best to cull frigid or constitutionally impotent males, independently of their genealogical value, as they may carry more serious genetic deficiencies; we support this view. (b) Semen control The technique of microscopic analysis of semen has not changed much from that recommended in past years. The use of bromine phenol-blue dye, which we modified and tested some years ago (Bonadonna & Olgiati, 1953) has been widely found to be preferable to any other method for ease of reading. The electron-microscopic analysis of spermatozoa, started by us in 1953, enables one to identify fine cellular structure and thus to determine the physiological state of the spermatozoa (Bonadonna, 1967). Investigations of particular interest have been made to detect-through light and electron-microscopic analysis-structural or histological change in the apical portion of the acrosomal area of the spermatozoan, which is a fragile region, in order to eliminate the action of hyaluronidase in vitro, with consequent influence on fertilizing ability (Bonadonna et al., 1968).

A WORLD SURVEY OF ARTIFICIAL BREEDING

(c) Diluents Many innovations have been made in the diluents used, mainly simple modifications in the quantities of the common diluents (sodium citrate, some amino acids and sugars, egg yolk, antibiotics). Less common substances have also been investigated, such as tris buffer (2-amino-2-hydroximethyl-1,3propanediol), which was tested by Nishikawa and is now being used, it seems successfully, by American investigators. An interesting finding is the relative tolerance of spermatozoa to carbonated waters and even, although to a less extent, to spring water (Bonadonna et at., 1964). Further results have recently been reported on the use of tomato juice and egg yolk in the freezing of bull semen by Chieffi and co-workers (1966). Vegetable juices were also tried successfully by us in 1964. The use of so-called synthetic diluents, mostly prepared in the dry state, is spreading. They generally consist of homogenized powdered milk with the addition of other ingredients such as amino acids, sugars, egg lipoproteins, mineral salts-chiefly potassium-and antibiotics and tranquilIizers. The liquid diluents Seminan and Neoseminan, prepared in Japan by Nishikawa, are now well known. A new diluent recently studied in New Zealand and being used for experimental purposes in Great Britain is Caprogen. Other diluents in the form of powders are prepared in the U.S.S.R. frem the formulas of Milovanov & Sokolavskaia (1968), and in Hungary (the Androsol diluent). A synthetic diluent in powder form which is mostly used in France is UNIDYL-J4. The dry diluents are generally dissolved in twice-distilled water with the addition of egg yolk. The possibility has arisen of preserving semen as a powder by lyophilization, and trials have been made in various countries, but still without success. Investigations in Texas by M. A. Brown and H. Faris (personal communication) have not yet yielded useful results, since not more than 5 ·82 per cent of the spermatozoa present could be reactivated. ( d) Techniques if innoculating semen The rectal method (also called the Anglo-American system) is still the one most used. The use of a differently shaped speculum is still rather widespread in some countries, mostly in Eastern Europe, but differences in fecundity and ease of use are negligible. The most important points are: (i) the dose of semen inoculated (generally from 0 . 5 to I m!. ), which must be related to the content of living and fertilizing spermatozoa of each dose (independent of the volume of semen). According to various authors this should be from IO to 25 million spermatozoa. (ii) Ensuring that insemination can really occur in ruminants, and not that spermatozoa only reach the cervical canal. Instruments have been designed everywhere for insemination which have been proved both efficient and cheap. Conservation if semen by freezing This method came into fashion recently and is spreading in all countries. Conservation of semen in liquid nitrogen at - 196°c is gradually replacing that (e)

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in carbon dioxide ice at - 79 °c, where liquid nitrogen is available. The use of liquid air (at about - 130°C) has been abandoned since it proved to be dangerous. The plant necessary for freezing semen in liquid nitrogen is expensive; the relative expense, however, is compensated for by the longer conservation of semen, by the possibility of sending it long distances and forming large semen stocks, which is important when matings are seasonal, and flexibility in the limits of using semen, which in practice is the most important advantage. The manufacture of suitable instruments and containers has been standardized everywhere by the major firms and is being improved and simplified. However, the freezing method is a rather delicate and complicated technique and must be carried out by skilled technicians, otherwise the consequences could be serious. It is difficult to forsee the extent of this possibility, but death of spermatozoa and failure of conception could be the least dangerous results, being immediate effects. A decrease in the DNA content of spermatozoa after freezing has frequently been shown (Hanada, 1965; Bonadonna & Fornaroli, 1966; Baicoianu & Dimoftachi, 1966, 1968). This would cause no anxiety biologically if it could be attributed only to progressive death of spermatozoa, but it is not yet possible to exclude an influence on the genetic material due to ageing of semen, since the mechanism of the prolonged survival and fertilizing capacity of spermatozoa under in vitro conditions is unknown. The storage of frozen semen is an important and much debated aspect of semen cryotechnique and can be carried out as foHows. (i) In ampullae or tubes of neutral glass, flame welded. This is till the most used system in many countries. Generally the glass tubes are 2-3 ml. in volume and contain I ml. offrozen semen. Glass tubes have recently been replaced by plastic tubes manufactured in Europe and America, with various plugging systems. (ii) In cylindrical plastic tubes, 1-1'5 ml. in volume, or in paillettes. The latter were suggested by Sorensen in Denmark (1940) for storing frozen semen and were also tested by us at that time. Semen stocks in paillettes were set up by R. Cas sou in France a few years ago; the method is being adopted-apparently with success-in many countries, particularly in Europe. (iii) In pellets, also called tablets, grains, or frozen drops. This method was devised in Japan and presented at the International Congress in Trento in 1964 (Nagase & Graham, 1964; Nagase & Niwa, 1964). Pellets are prepared by dropping semen on blocks of carbon dioxide ice or on plates exposed to liquid nitrogen vapour. By this latter system Seifert & Beller (1966) in Peru could freeze, with good results, stallion semen (Nagase et at., 1966); good results were also obtained with paillettes. According to investigations by the Danish authors Rasbech (1960) and Adler (1960) using containers other than ampullae, the fertilizing capacity of frozen spermatozoa would be 5-10 per cent lower than that of semen kept at + 5 DC. The paillettes system is less expensive but would give a 5 per cent lower conception rate. The use of pellets is even cheaper, but according to Medling (1966) would

A WORLD SURVEY OF ARTIFICIAL BREEDING

produce a 35 per cent lower conception rate than semen kept at +5 °c. The Cooperative d'Insemination Artificielle de l'Aigle (Bilan d' Activite, 1965-66), where there were 202,721 A.I. cows (184,803 of Norman breed) with 67 ·61 per cent "non-returns" to heat, reported an average annual production of 12,000 paillettes per bull, with a maximum production of30,000 and a total of 650,000. The use of 0'5 ml. paillettes gave better results (from 69'54 to 69'93 per cent of "non-returns") than I ml. paillettes. According to the same source ofinformation, the world total number of inseminations made by paillettes is about 15 million, mostly with frozen semen. However, the use of pellets is assuming greater importance and spreading in both Western and Eastern Europe and in South America. The problem of possible contamination of semen either during collection or in the later operations was early considered by various investigators and by ourselves. Of particular interest are the observations of Russian and French authors (see Bonadonna, 1967), confirmed by our recent investigations (Bonadonna et at., 1968). The possibility of microbial viability and pathogenicity in the frozen semen is a serious problem. The foot-and-mouth disease virus-the elimination of which through the semen occurs during the first 5 days of the infection (Gierl",ff & Jakobsen, I960)- in vaccinated animals is thought to persist in the frozen semen, which becomes a possible means of transmission. Many investigations confirm the survival of the pathogenic agents of brucellosis and vibriosis in frozen semen (even treated with penicillin and streptomycin, as Adler reported in 1966), as well as of trichomoniasis (Arache, 1964; Bonadonna, 1965). FRO ZEN SEMEN BANKS AND INTERNATIONAL EXCHANGES

The early frozen semen banks were established in West Germany (at Munich) and Canada and later in Holland, as reported in 1957; they are now found in many countries. In Australia and in New Zealand they meet the requirements of seasonal reproduction (Bonadonna, 1963). Organizations for the production and distribution of semen in the U.S.A. and Canada are equipped with large plants and thus contribute considerably to international exchanges of semen. The U.S.A. has the largest distribution area, which is world-wide, and operates particularly in Latin America and Australia. In 1965 the U.S.A. supplied thirty-seven countries with the semen of seventeen different breeds; in 1966 they imported into Stonwal, Oklahoma from France the semen of bulls of the Charolais breed, thus contravening the stringent rules for the first time (A.I. Digest, NO.9, 10). On the whole, however, international exchanges are less extensive than has been thought. Great Britain exports a certain number of doses of frozen semen annually, chiefly to the East and to East and South Africa. At "Expo 67" held in Montreal in 1967, an A.I. calf generated by frozen semen collected 10 years previously was shown; in 1966 a calf had been generated by frozen semen after I I years' conservation.

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HEAT SYNCHRONIZATION

Oestrus synchronization in groups of females to be artificially inseminated is important for the largest herds of some species (sheep, goats and pigs) in certain districts, for economic reasons. According to some authors, heat synchronization would also be very useful in cattle. However, it is particularly important in sheep and goats, to enable the full use of time and pasture (bearing in mind the seasonal transfer of herds,) in order to obtain higher outputs (lamb for meat, goat milk for the industry and for direct consumption). In pigs, oestrus synchronization may help in the boar progeny test, as it is thus possible to inseminate a larger population of sows at one time. BULL SELECTION CRITERIA

In countries where animal husbandry is well developed, and especially in those efficient cooperative breeders' organizations, the selection of bulls for A.1. is rigid and the general criterion is to use progeny-tested bulls only. This holds for dairy and beef breeds, and for those of mixed type. The young bulls to be tested genetically are generally used to inseminate 500 cows, so that at least forty daughters may be available. Later they may be used in given districts, as is done in England, or in so-called "commercial" herds. This principle implies a suitable organization for stocking the semen of valuable bulls until their genetic testing is completed. In many countries progeny testing of dairy bulls is completed with individual morphological appraisal and records of each male's progeny as to increases in weight, yield at culling, proportion of beef to fat, skeleton weight, and so on. It is best to combine the progeny test relating to production with that offecundity, assessed through the sexual behaviour of both male and female offspring. This criterion is fundamental not only to the "individual" and "family" yield, but also to keeping average fecundity in populations and breeds at the most suitable levels. AVERAGE FECUNDITY IN ARTIFICIALLY INSEMINATED CATTLE

The criteria suggested for assessing fecundity include the rectal method of diagnosis 30-40 days after insemination; the so-called non-return to heat after 6090 days (or after 90- I 20 days); and the percentage of calves born. Diagnosis by the rectal method is used in only a few countries (in Eastern and Western Europe) but it should be adopted when statistics are collected by sampling or when assessing the actual fertilizing capacity of bulls or female sexual health. From the economic point of view the significant criterion is that of the nu,mber of births, which is the one used in the Scandinavian countries. Diagnosis on the basis of non-return to heat is the most popular method, as it is the easiest, particularly where technicians rather than veterinarians are operating, but it is not an infallible method. Fecundity is often reduced by the lack of skill of the technicians in preparing or inoculating semen. Average fecundity at first insemination should be from

A WORLD SURVEY OF ARTIFICIAL BREEDING

53 to 60 per cent, based on diagnosis at 30-40 days made by the rectal method. It is about 65-70 per cent when estimated by the non-return to heat 60-90 days after insemination. The American Breeders Service reports an average of 72 per cent non-returns after frozen semen inoculation in 6 million cows from 1955 to 1963; this is a very high average rate. In some developed countries, such as Denmark and Holland, the fecundity rate for the first insemination is surprisingly low (from 60 to 66 per cent). However, this rate roughly corresponds to that obtained by the rectal method of diagnosis. From investigations made by us in the well-watered district near Milan-that is, a particularly difficult breeding area-the fecundity rate at first insemination was from 48'3 to 73 per cent and the final rate in the year was from 84'5 to 96'7 per cent, by the rectal method. An average of 1'5-1'7 in the pregnancy/insemination ratio is satisfactory, but if the ratio increases to 2-2 '5, secondary sources of failure are operating. Veterinarians, animal husbandry experts and breeders are still inclined to relate failures in conception to unspecified infectious causes; less frequently failures are referred to environmental factors and particularly to feeding, and to the high density of animals on a given area (Barigozzi, 1966; Bonadonna, 1967). The main problems concern genetic factors (both hereditary and non-hereditary) ; embryonic and foetal mortality; still-births and neonatal deaths; chromosomal and morphologicalfunctional abnormalities; infertility for immunological reasons. Because of these possibilities, the fecundity progeny test is more important than the production progeny test, although the latter is traditionally the one considered. Research into these problems is needed that will require a new direction in animal husbandry. RESPONSIBILITY FOR INSEMINATION

In most countries inseminations are carried out by non-veterinary staff (technicians) working under the supervision and responsibility of veterinarians, directors of A. I. centres, or public health officers. In Italy and in many other countries, it is required by law that inseminations be carried out by veterinarians; however, with some exceptions, ordinary technicians may also assist. Countries are free to determine the level of competence that they require for their particular needs, but the general principle being followed is that of adequate training of technicians and veterinarians, but without the requirement of clinical competence in technicians. The exception to this is the grade of "sanitary attendants" or "veterinary assistants", found in some countries, e.g. the U.S.S.R. CONCLUSIONS

Artificial insemination has been adopted in animal breeding throughout the world to increase production and to speed up the rate of improvement and the spread of uniformi ty in animal stocks. It has achieved successful results of some importance for the most developed countries and for the developing ones. The practice of artificial insemination needs to be closely controlled, on the basis of a knowledge of genetics and hygiene. This is why, now that operating tech-

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niques are easier, it becomes increasingly necessary for A.I. to be controlled by specialized institutes and by experienced technicians. The field of reproduction is the most important in animal husbandry, but also the most complex and the least easily understood. REFERENCES ADLER, H. C. (1960). Aarsberetning, 243-247. ADLER, H. C. (1966). Aarsberetning, 149-154. ARACHE, J. (1964) . Methode de Conservation du Sperme de Taureau a Tres Basse Temperature. Utilisation Pratieue de l' Azote Liquide. Alfort. Paris: BAlCOlANU, C. & DIMOFrACHE, C. (1966). Rev. zoot. med. Vet. 7, 44. BAlCOlANU, C. & DIMOFTACHE, C. (1968). Zootee. Vet., 23, 105-114. BARlGOZZI, C. (1966). Metodi Modernissimi per Accertare la Sterilata. Corriere della Sera. BONADONNA, T. (1963). L'Allevatore, 19, Nos. 15, 16, 17. BONADONNA, T . (1965). Gazzetta Veterinaria, 3-4, 6-11. BONADONNA, T. (1967). Ace. Naz. agrie. Annali, (series Ill), 77. BONADONNA, T. (1967). Progressi ed aspetti di attualita nella fecondazione artificiale degli animali. Atti del Corso di Aggiornamento di S. Miniato, Pisa, 1966. pp. 259- 290. BONADONNA, T. (1967). Annali Ace. agrie. Torino, 129. BONADONNA, T. (1967). Zootee. Vet., 229, 212-224. BONADONNA, T. (1967). Zootee. Vet., 22, 311-359. BONADONNA, T. (1967). Bull. Agrie., 101 (23). BONADONNA, T., DUMITRESCU, I., NESTl, M. G., VERMA, B. N. & CESARlNl, R. (1968). Clin. Vet. (in press). BONADONNA, T . & FORNAROLI, D. (1966). Zootee. Vet., 219, 204- 206. BONADONNA, T., FORNAROLl, D., EFSTATlON, L. & POZZI, G. C. (1964). Rev. Ciene. Veter., 59, 390. BONADONNA, T. & OLGIATl, L. (1953). Zootee. Vet., 7,195-199. CHIEFFI, A. et al. (1966) . VCongreso Panamericano de Medineca Vet.y Zoot., Caracas, Venezuela. GIERL0FF, C. C. & JAKOBSEN, Kj. F. (1960). Aarsberetning, 293. HANADA, A. (1965). Jap. J. Anim. Reprod. 3, 91 -94. MEDING, J. H. (1966). Aarsberetning, 179-183. MILOVANOV, V. K. & SOKOLOVSKAJA, I. (1968). Zootee. Vet. 23, 3-77. NAGASE, H. & GRAHAM, E. F. (1964) . Proceedings of the Vth International Congress on Animal Reproduction and A.I., 4, 404- 409. NAGASE, H. & GRAHAM, E. F. (1964). Proceedings of the Vth International Congress on Animal Reproduction and A.I., 4> 387- 391. NAGASE, H. & NIWA, T. (1964). Proceedings of the Vth International Congress on Animal Reproduction and A.I., 4> 410-415. NAGASE, H. et al. (1966). Jap. J. Anim. Reprod., 12, 48-51,52-57. RASBECH, N. O. (1960) . Aarsberetning, 243-247. SEIFERT, H. & BELLER, K. A. (1966). VCongreso Panamerieano de Medeeina Veterinariay Zootecnia, Caracas, Venezuala . SORENSEN, Ed. (1940). Medlemsbl. danske Dyrlaegeforen, 23,166-169.