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C. Polge

Reproductive efficiency is one of the most important components in the economy of animal production and it is well recognized that it can be influenced by many factors affecting different areas of the reproductive process. Fertilization is just one of the essential links in the whole chain of reproductive events leading to the production of offspring, but it is the link most closely associated with the efficiency of breeding by means of artificial insemination. Different techniques used in A.I., especially those concerned with methods of insemination or with the dilution and storage of semen, can therefore best be assessed by their effects on fertilization and these aspects will be emphasized in the present paper.

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I. WILMUT and C. POLGE

Summary.

Three experiments have been carried out to investigate the mechanism by which glycerol reduces the fertilizing capacity of boar spermatozoa during storage, and any relationship between this effect and the relatively poor conception rates obtained with deep-frozen boar semen.

The loss of fertilizing capacity in the presence of glycerol was shown to be temperature-dependent, Fertilizing capacity was significantly reduced after 6 hr of incubation at 20°C in the presence of 5 or 10% glycerol, although there was no detectable change in motility or morphology of the spermatozoa. By contrast, there was no effect on fertilizing ability after storage for 6 hr at 5°C or for 30 min at 20°C in the presence of these concentrations of glycerol. Removal of the seminal plasma and a large proportion of the free cytoplasmic droplets did not prevent the reduction in fertilizing capacity. When semen was frozen and thawed, attempts to minimize the exposure of spermatozoa to glycerol did not lead to fertilization when the thawed semen was inseminated through the cervix. It is suggested that at ambient temperature glycerol causes damage to boar spermatozoa which is exacerbated by the stress of freezing and thawing.

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C. POLGE and P. DZIUK

In the pig, as in most mammalian species examined, the eggs are normally ovulated as secondary oocytes (Pl. 1, Fig. 1); the first polar body has been emitted and the chromosomes are arranged at metaphase on the second maturation spindle (Corner, 1917; Spalding, Berry & Moffitt, 1955; Pitkyanen, 1961). Hancock (1961), however, found three out of 1677 normally ovulated pig eggs which were primary oocytes and Spalding et al. recovered one immature egg after induced ovulation. On rare occasions, primary oocytes have also been recovered from the Fallopian tubes of rats and mice, but these immature eggs do not appear to be fertilizable even though spermatozoa may pass through the zona pellucida (Austin & Braden, 1954). During the course of experiments on the fertilization of pig eggs following induced ovulation (Dziuk & Polge, 1965), 1472
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W. D. Booth and C. Polge

Summary.

Five true hermaphrodite pigs and two male pseudohermaphrodite pigs were studied. A 38XX sex chromosome constitution was found in peripheral leucocytes of three true hermaphrodites and in one male pseudohermaphrodite; XX/XY mixoploidy was present in the leucocytes of the remaining male pseudohermaphrodite. The occurrence of C19 steroids, including 16-androstenes, in the testicular tissue and submaxillary gland of intersex pigs was of a similar pattern to that found previously in mature boars, and masculinization of the genital tract was related to the amount of testicular tissue present. It is postulated that in the absence of germ cells in the testicular tissue of intersex pigs the Sertoli cells may be involved in the metabolism of dehydroepiandrosterone to 5-androstenediol, a possible testosterone precursor in the pig. The high levels of 16-androstenes found in the submaxillary gland of intersex pigs indicates that these steroids are responsible for `boar taint' in these animals. In contrast to the boar, no consistent relationship was found between the occurrence of C19 steroids and the degree of masculinization of the submaxillary gland; it is postulated that the predominantly female genetic constitution may have affected the response of the salivary gland to androgen.

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R. D. BAKER and C. POLGE

Summary.

Boar spermatozoa fertilized only a small proportion of the follicular and ovulated eggs that were transferred into the uterus of artificially inseminated gilts. The possibility that spermatozoa had entered the oviduct or were influenced by tubal secretions before penetration cannot be excluded. Sperm penetration in utero was not observed when oviducts were ligated at the uterotubal junction before transferring eggs into the uterus.

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H. D. Guthrie and C. Polge

Treatment of cattle with various doses of prostaglandin (PG) F-2α by subcutaneous injection (Lauderdale, 1972) or by nonsurgical intrauterine injection (Rowson, Tervit & Brand, 1972; Liehr, Marion & Olson, 1972; Louis, Hafs & Morrow, 1972) induces luteal regression and allows oestrus to occur if initiated after Days 3 or 4 of the oestrous cycle. The oestrous cycle of the mare can also be shortened by administration of PGF-2α (Douglas & Ginther, 1972). Because the presence of the uterus is required for normal cyclic function in the pig (Anderson, Bland & Melampy, 1969), the effects of a synthetic analogue of PGF-2α, ICI 79,939, on luteal function and oestrus in gilts was investigated.

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H. D. Guthrie and C. Polge

Treatment of cows or mares with prostaglandin (PG) F-2α as early as Days 4 or 5 of the oestrous cycle causes regression of corpora lutea (CL) and results in oestrus 2–4 days after treatment (Lauderdale, 1972; Rowson, Tervit & Brand, 1972; Douglas & Ginther, 1972; Allen & Rowson, 1973) However, the CL of pigs do not regress in response to PGF-2α until Days 11–12 of the cycle (Gleeson, 1974; Guthrie & Polge, 1976). Because the secretory activity of the CL in the pig declines rapidly by Days 14 or 15 of the cycle, the effective period for synchronization of oestrus by PGF-2α treatment is too short to be of practical use. Exogenous gonadotrophins induce the formation of accessory CL at any stage of the oestrous cycle of the pig (Neill & Day 1964; Day, Neill, Oxenreider, Waite & Lasley, 1965; Caldwell, Moor, Wilmut, Polge & Rowson, 1969) and the CL formed on Days 8, 10 or 16 delayed oestrus for at least 15 days (Caldwell et al., 1969). We therefore investigated the possibility of synchronizing oestrus in gilts by inducing accessory CL to delay oestrus and ovulation and then injecting PGF-2α analogues to regress the accessory CL at a predetermined time.

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B. N. DAY and C. POLGE

Polyspermy can be produced experimentally in pigs by delayed mating, which results in the fertilization of aged eggs (Pitkjanen, 1955; Thibault, 1959; Hancock, 1959; Dziuk & Polge, 1962; Hunter, 1967). A high incidence of polyspermy has also been observed in pigs following induced ovulation and insemination during the luteal phase of the oestrous cycle (Hunter, 1966). These observations suggest that the endogenous level of progesterone at the time of fertilization might affect the block to polyspermy in pig eggs, since, under both conditions cited, developing or fully functional corpora lutea would be present in the ovaries at the time of sperm penetration. The present study was conducted to determine the frequency of polyspermy in pigs injected with progesterone at various intervals before ovulation and fertilization.

The experimental animals were thirty-six mature, crossbred, Large White × Essex,

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H. D. Guthrie and C. Polge

Summary. Oestrus was induced 4–7 days after treatment with Cloprostenol (ICI 80,996) in gilts between 12 and 40 days pregnant. Fertility at this synchronized oestrus was good (85%).

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W. F. Rall and C. Polge

Summary. Mouse embryos (8-cell) fully equilibrated in 1·5 M-glycerol were cooled slowly (0·5°C/min) to temperatures between − 7·5 and − 80°C before rapid cooling and storage in liquid nitrogen (−196°C). Some embryos survived rapid warming (~500°C/min) irrespective of the temperature at which slow cooling was terminated. However, the highest levels of survival of rapidly warmed embryos were observed when slow cooling was terminated between − 25 and − 80°C (74–86%). In contrast, high survival (75–86%) was obtained after slow warming ( ~ 2°C/min) only when slow cooling was continued to − 55°C or below before transfer into liquid N2. Injury to embryos cooled slowly to − 30°C and then rapidly to −196°C occurred only when slow warming (~2°C/min) was continued to − 60°C or above.

Parallel cryomicroscopical observations indicated that embryos became dehydrated during slow cooling to − 30°C and did not freeze intracellularly during subsequent rapid cooling ( ~ 250°C/min) to − 150°C. During slow warming (2°C/min), however, intracellular ice appeared at a temperature between −70 and − 65°C and melted when warming was continued to − 30°C. Intracellular freezing was not observed during rapid warming (250°C/min) or during slow warming when slow cooling had been continued to − 65°C.

These results indicate that glycerol provides superior or equal protection when compared to dimethyl sulphoxide against the deleterious effects of freezing and thawing.