These experiments investigated the effect of freezing on mitochondrial function in ram sperm, the effectiveness of current freezing procedures in protecting mitochondria, and the role of mitochondrial respiration in cervical penetration and transit by ram sperm. Only sperm with functioning mitochondria (assessed by rhodamine 123 staining) after freezing and thawing were motile in a viscous medium (P < 0.05). A simplified rhodamine 123 uptake assay was developed to monitor sperm mitochondrial function. The results of this procedure were highly correlated (r2 = 0.98) with the proportion of damaged sperm in the semen sample. A semen freezing procedure commonly used by industry was compared with newer methods, and with freezing without cryoprotectants. None of the freezing protocols produced sperm with higher post-thaw levels of mitochondrial integrity than unprotected sperm. Merino ewes were inseminated with semen treated with metabolic inhibitors. Glycolytic inhibition did not affect fertility. Mitochondrial inhibition reduced fertility in cervically (P < 0.05), but not laparoscopically inseminated ewes. It is concluded that mitochondrial respiration plays an important part in penetration of the cervix by ram sperm. Mitochondrial injury during freezing is likely to be implicated in the poor fertility of frozen ram semen used for cervical insemination.

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Historical BackgroundCurrent interest in the possibility of predetermining the sex of human or animal progeny is only a recent manifestation of an age-old phenomenon. The antiquity of the subject is demonstrated by Talmudic references to coital methods of sex preselection, although their interpretation remains obscure (Rosner 1979).The possibility of choosing the sex of a child was also considered by fifth century Greeks. Despite their belief that supernatural forces designated fetal sex, selective infanticide was used to dispose of children with a variety of undesirable characteristics, including inappropriate sex [Cyrus the Great (Heredotus, Book I) and Oedipus]. As an alternative, parents were given access to a number of pre-natal sex selection measures (reviewed by Levin 1987).Hippocrates, for example, proposed that males developed more readily in the right side of the uterus, and females in the left. Appropriate sexual practices were believed to ensure deposition of semen in the correct portion of the female tract, followed (at least in theory) by development of a fetus of the desired sex. Alternatively, ligation or, in extreme cases, removal of the right testis (believed to produce males) or left testis would result in dispatch of the correct type of sperm. Other techniques relied upon manipulation of such factors as the geographical alignment of the nuptial bed, or the removal or retention of boots prior to sexual activity. Even Aristotle, whose belief in semen as the controlling factor in fetal development approached the absolute, attributed sex determination to the relative activity of the parents during intercourse. The popularity of such folkloric methods was maintained into the 20th century, by which time a variety of dietary, sexual and surgical techniques was also included.Such practices were based less upon empirical evidence than on the assumption that sex was determined 'not by inheritance but by the combined effect of external conditions' (Wilson 1896, cited by Farley 1982. This axiom was challenged by the observation that Mendelian genetics provided a possible mechanism for the inheritance of sex at the time of fertilization and the eventual realization that sex was determined by the chromosomal complement of the fertilizing sperm (reviewed by Farley 1982). Recognition of the fact that (in mammals) fertilization by X chromosome-bearing sperm produces females, and Y sperm males, has resulted in most of the scientific 20th century attempts at preconceptual sex selection involving alteration of the X: Y ratio in a sperm population, either in vitro prior to artificial insemination, or in the female tract.Impetus for sexing human sperm was provided by the desire to reduce the incidence of sex-linked genetic disorders. Improvements in artificial breeding technology and the demand for more efficient agricultural production has, however, led to increasing interest in predetermining the sex of the offspring of domestic livestock. The separation of X and Y

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