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Recent advances in the deep-freeze preservation of ram semen

1974; African Journals OnLine; Volume: 4; Issue: 2 Linguagem: Inglês

2221-4062

D Visser,

Reproductive biology and impacts on aquatic species

Research on the frozen storage of ram semen has been going on for nrore than 20 years and in general followed the same lines as with bull semenln an excellent review by Ughtfoot ( 1969) most aspects of the freezing procedures were discussed fully, and detailed assessments were pres€nted on the effects of diluent composition and nrethods of dilution, cooling, freezing and thawing. A brief discussion of sonp of these procedures is presented here as an introduction to the review of fertility results with frozen ram sperrnatozoa Thc drluents commonly ued are isotonic to slightly hypertonic media and the inclusion of sugars, notably those of higher molecular nrcight, was found to have beneficial effects. The commonly used cryoprotective agent, as for bull sernen, is glycerol. Dlution of the semen at 3@C with the glycerol+ontaining diluent (one-otep dilution) can be done suc*ssfully and this has considerable practical ad%ntages over the rnethod of addition of the glycerol+ontaining fraction at 5oC (two-step dilution). Lower rates of dilution (l:2 to l:4) of semen are generally reported to be nrore suitable, but this can be influenced by factors such as the composition of the diluent and the rnethod of freezing. Results on cooling rates indicated that slow cooling to 5oC orer one to two hours is required and that relatirely short periods of equilibration at 5oC gave satisfactory ressults with ram serpn. Ram scnren can be frozen successfully in amp6ules, straun (paillettes) or in pellet form, and choice of freezing method seemed dependent largely on individual preferences and needs. It is noteworthy though that the straws and ercn more so pellets, offer substantially grettet utilization of storage space in the liquid nitrogen containers than do ampoules. Two important considerations should further be borne in mind when the semen is frozen in pellet form. First, tfuough lack of a sealed container, bacterial contamination in liquid nitrogen is possible and the probable event of migration of spermatozoa between semen-pellets of different sires (Merkt, Weitze & Lorrmann, 1967) may raise doubt on the parenthod of the p'rogeny obtained. In the second place, pelleted semen makes pos. sible the we of a thawing solution which can hare a substantial effect on the recovery rate on thawing, as well as on the survival of cells during post-thawing incubation. It further follows that the use of a thawing solution implies further dilution of the semen and reconcentration (by centrifugation) before insemination is necessary to obtain satisfactory fertility. The findinp of the reports by Ughtfoot (1969) were further substantiated by results of Lightfoot & Salamon (1969a, b), Salamon & Lightfoot (1969) and Salamon & Brandon (1971). It was, nevertheless, indicated by these authors that numerous factors of diluent composition interacted with each other and with nrethods of dilution, cooling, freezing and thawing. It is therefore unwise to resrmrnend a certain diluting media when other freeze-thawing procedures are not specified. This complex influencc of various factors therefore explains to a great extent the wide variation and often total contradictron found in published results. The early research on the fertility of frozen-thawed ram sperrnatozoa has been reviewed by Emmens (1961), Emmens & Robinson (1962), Sadleir (1966), Iridl (1963) and Lightfoot (196!9). In the report by Ughtfoot a full summary of the literature up to that time is presented and the author also referred to the problem of evaluating some results where the authrrrs used unsatisfactory experimental designs and/or provided insufficient details on the conduct of experiments. Subsequently, Lopyrin (1969) also criticised the methods used by some Sorriet workers who claimed high fertility. When these claims were tested under strict supervision none of the authors could confirm their own results and obtain a fertility rate higher th^n 14.o after one inscmination. Evidently, this was due to the replacernent of fertile teaser rans by yasectomised rams. Thns, the possibility of occasional fertilization of ewes by natural mating has bcen excluded. ln the review presented here, which covers research since 1968, lack of essential details in reports was still encountered and this is evident in the sumrnary of the literature presented in Table l. Nevertheless, the fertility results following insemination with frozen-thawed ram semen improved markedly in the past five years. They are still lower than those obtained with fresh-diluted semen, but the important point is that these results are at least repcatable. ln the discussion that follows, these results will be examined under several headings relating to the most relenant procedures of fteeze-thawing and insemination. Thus, diluent composition is dealt with first, followed by dilution rate, freezing and thawing methods, insemination techniques, finally concluding with the effect of long-term storage on fertility.