4. Effects of nutrient imbalance on reproduction
MUTATIONS CAUSED BY FOLIC ACID DEFICIENCY
Pernicious anaemia appears to have been the first well-known human disorder shown to be associated with the production of chromosomal abnormalities and an increased human mutation rate. The knowledge that anaemia and other illness was associated with visible damage to the cell nucleus goes back to the 19th century. Fragments of the cell nucleus found within red blood cells were discussed in 1899 by Sebmauch of the Royal Pathological Institute of Konigsberg. His 20-item bibliography from 1865 includes many descriptions of the occurrence of these fragments in animals and humans associated with illness. Following a paper by Howell (1890), an American physiologist, these nuclear fragments became known as "Howell's bodies", or sometimes as Howell-Jolly bodies. Jolly (1905) being a French histologist who also described these microscopic particles. Although the appearance and even the number of these particles depends upon the techniques used for their study they are essentially the same particles described today as micronuclei.
More than half a century later Hutchison and Ferguson-Smith (1959) from the University of Glasgow published a paper on the significance of Howell-Jolly bodies which they had identified in 52 consecutive cases of pernicious anaemia. It was well-known by 1959 that pernicious anaemia could be produced by deficiencies of folic acid or of cobalamin (B12). Chromosomes were first described in 1873 and acquired their name in 1888 and their functions were broadly understood by the turn of the century. Hutchison and Ferguson-Smith appear, however, to have been the first to draw the separate lines of investigation together and to conclude that Howell-Jolly bodies were partly composed of chromosome fragments and were formed as a result of nutrient deficiencies. Hutchinson & Ferguson-Smith concluded:
"It seems probable that disordered nucleic acid synthesis resulting from B12 or folic acid I deficiency is the fundamental factor in the production of this abnormal behaviour at mitosis."
Potier de Courcy (1966) of the University of Paris showed that in rats folic acid deficiency slows down synthesis in the embryo of DNA, RNA and protein. Papers from a number of countries showed that there was an association between folic acid or cobalamin deficiency and chromosomal damage in somatic cells, generally blood cells (Astaldi et at., 1962; Forteza & Baguena, 1963; Kiossoglou et al., 1965; Heath, 1966; Menzies et al., 1966; Bottura & Coutinho, 1967). It was confirmed by these investigators that Howell-Jolly bodies or micronuclei could be produced by nutrient deficiencies, and that a range of chromosomal abnormalities could be produced by folic acid or cobalamin deficiency which were mutagenic at least in somatic cells. Heath (1966) reported that cobalamin and folic acid deficiency in cell culture increased chromosome breakage but without producing any characteristic, recognizable type of aberration. Menzies et al. (1966) found that cobalamin and folic acid deficiency caused "numerous morphological abnormalities" of chro mosomes. Hall and Davidson (1968) referred to the "widespread cytological dystrophy" caused by folic acid deficiency. A later paper noted that a folic acid antagonist could cause breakage of any chromosome and could cause single or multiple breaks or "total fragmentation or pulverization" (Mandello et al., 1984). Folic acid deficiency has been shown to cause nondisjunction in mice (Gates et al., 1981).
Stevenson (1978) described the effects of 12 folic acid analogues which behave like folic acid antagonists producing the symptoms of folic acid deficiency. Methotrexate and aminopterin are perhaps the best known of these folic acid antagonists. Stevenson showed, as would be expected, that 11 out of 12 of these compounds produced chromosomal abnormalities in human lymphocytes in cell culture. All eff00ects of these compounds could be prevented with tetrahydrofolic acid, the active reduction product of folic acid and also by thymine, one of the essential DNA bases requiring folic acid for its synthesis. The frequency of occurrence of micronuclei, or Howell-Jolly bodies, in the cells from normal individuals cultured in a medium deficient in folic acid or thymidine or both is shown in Figure 4.1. (Jacky et al. 1983).
The number of micronuclei is seen to have been 10 times higher in the medium deficient in both these nutrients which appear to be interchangeable in this special context. Similar studies have been published by Reidy et al. (1983) of the US Center for Environmental Health, Georgia. The frequency of occurrence of chromosomal breaks in the cultured cells of 25 normal individuals is shown in Figure 4.2 and of chromosomal gaps in Figure 4.3. In both cases the frequency of chromosomal abnormalities is compared for cells cultured in media containing and not containing folic acid. The authors comment that the variations between people in their chromosomal breakage rates may be caused by their different folic acid status, but also by other differences in the composition of their blood plasma or intracellular fluids. It is apparent that folic acid deficiency increases mutation rates which would be expected to cause infertility when it seriously affects germ cells. Mathur et al. (1977), reported on the effects of folic acid deficiency produced by aminopterin, one of the folic acid antagonists, on spermatogenesis in male albino rats. Aminopterin was used in doses too low to produce any overt toxic effects. Spermatogenesis was affected within 7 days and changes in blood cells and bone marrow in 15 days. The authors commented:
"This finding indicates that the testis is even more sensitive to folic acid deficiency than is hemopoietic tissue."
The folic acid deficiency resulted in the production of chromosomal abnormalities and a progressive reduction in the number of sperm cells at all stages of development as the deficiency continued. Folic acid antagonists used medicinally have been reported to cause large reductions in sperm counts in male patients (Sussman & Leonard, 1980). Adams (1958) had reported in the Scottish Medical Journal that involuntary infertility was a "striking feature" common to a series of women patients with pernicious anaemia. A number of studies followed of infertility in men associated with cobalamin deficiency. Watson (1962) reported the association of low seminal cobalamin with sterility and morphological abnormalities of spenn. This association of sperm quality and motility with cobalamin concentration had at this time already been reported in the veterinary literature, for example, as influencing the fertility of bulls (Busch, 1954). Sharp and Witts (1962) of the Radcliffe Infirmary, Oxford, pointed out that damage to sperm in men predated symptoms of anaemia suggesting that germ cells are more sensitive to deficiency of cobalamin than the cells of the bone marrow:
"It is well known that pernicious anaemia may be present in a latent form for some years before giving rise to symptoms of anaemia. The vitamin B12 in the serum should therefore always be estimated when unexplained impairment of spermatogenesis is found."
Adams (1958) had reported that sterility associated with subclinical anaemia in women preceded overt symptoms by a long period and this was confirmed by Smith (1962) who concluded:
"It seems desirable to screen all patients, both male and female, with unexplained sterility for subclinical evidence of Addisonian anaemia."
Such screening depends upon definitions and acceptance of "subclinical evidence" of a damaging deficiency. Megaloblastic anaemia has been regarded as the definitive evidence of cobalamin and folic acid deficiencies. However, deficiencies of these nutrients have been reported to be associated with pathological consequences without being sufficiently severe to produce megaloblastic changes in blood cells. Thus Tomaszewsky et at. (1963) published data showing that semen cobalamin concentrations associated with oligospermia and azospermia were below average but were not as low as would be expected for patients with overt megaloblastic anaemia. The greater sensitivity of germ cells compared with bone-marrow cells to moderate deficiencies of cobalamin could explain the sterility that precedes megaloblastic anaemia by months or even years. Other body systems including the immune system and central nervous system are affected by marginal deficiencies of both cobalamin and folic acid insufficient to produce anaemia.
Steinberg et at. (1983), of the Universities of Minnesota and Washington used radioactive labelled folic acid cells cultured in the laboratory. A critical level of intracellular folic acid concentration was found of 0.1 ng/l 06 cells below which DNA replication and cell division cease. Only below this level Effects of Nutrient Imbalance on Reproduction do "bizarre multinucleate cells" and megaloblastosis become apparent. However intracellular concentrations above this threshold in the range 0.1 to 0.5ng/106 cells are associated with abnormalities of DNA synthesis. Megaloblastic anaemia is seen to be evidence only of extreme folic acid deficiency, and there are less serious levels of deficiency that still interfere with DNA synthesis. The morphological criteria of deficiency are much less sensitive than biochemical criteria. In more usual units, not used by Steinberg, there is a risk of megaloblastic anaemia at intracellular concentrations below about l4Ong/ml, but of some interference with DNA synthesis up to much higher concentrations d0iminishing with increase in concentrations up to 3 or 4 times this figure. The l4Ong/ml is the usually accepted "high risk" threshold (Sauberlich et al., 1977).
NUTRITIONAL DEFICIENCY DURING PERIODS OF HIGH SUSCEPTIBILITY
In the 1940s and 1950s there were three important research centres studying the effects of nutritional deficiencies on reproduction, in France at the Medical School of the University of Paris, in Switzerland at the University of Geneva and in the USA at the University of California. All these centres showed that in female animals deficiencies of essential nutrients could affect fertility and the health of offspring. Watteville et al. (1954) of the University of Geneva showed that a deficiency of any one of the four B vitamins thiamin, riboflavin, pyridoxine or pantothenic acid beginning 13 days before mating reduced the fertility of rat dams by up to 80 per cent and beginning 28 days before mating by 100 per cent. By 1951 it had already been shown that a deficiency of vitamin A could cause both infertility and congenital malformations (Hale, 1933). It had also been shown that a deficiency of folic acid could cause infertility and malformations (Nelson & Evans, 1949; Giroud & LefebvresBoisselot, 1951). Ross and Pike (1956) of Pennsylvania State University showed that rats deprived of pyridoxine before mating produced pups of low birthweight even when pyridoxine was restored to the diet immediately after mating. They concluded:
"The data indicate that pyridoxine in the diet before mating is as important as pyridoxine in the diet during gestation; giving further support to the hypothesis that the condition of the matemal organism prior to the inception of pregnancy plays a critical role in the course of pregnancy and its outcome."
Nelson and Evans (1955) of the University of California found that thiamin deficiency initiated 11 to 15 days before mating caused 83 per cent of embryos to be resorbed as illustrated in Figure 4.4. If the deficiency was initiated earlier still most animals had no implantations. Thiamin deprivation before mating resulted in low birthweight among surviving pups of the deficient dams as illustrated in Figure 4.5. It was not very clear in any of these experiments just when the induced deficiencies had their physiological effects, but the effects were most marked when the nutritional deficiency began before mating and if begun early enough it always inhibited ovulation. If the deficiencies were initiated somewhat later nearer mating 0ovulation proceeded but implantation was inhibited, or if implantation proceeded the embryo could be malformed or be resorbed.
The deficiency needed to produce these results was modest. Riboflavin deficiency was reported to cause malformations in rats by Warkany and Schraffenberger in 1943. The same authors said in a later paper (Warkany & Schraffenberg, 1944):
"We are dealing in these experiments with a borderline deficiency ... Abnormal offspring appear when the riboflavin of the blood reaches a certain critical level. A reduction of riboflavin below the critical level leads to sterility and embryonic death, while an increase beyond this level results in the birth of normal young."
Warkany was a pioneer in the study of causes of congenital malformations (Warkany, 1971). Giroud et al. (1949, 1950, 1952) pursued research on riboflavin deficiency at the Medical School of the University of Paris. Giroud failed to produce any malformations by depriving female rats of riboflavin beginning only from or after mating, but a high percentage of resorbed embryos and malformed young were produced when riboflavin deprivation began 11 to 22 days before mating as shown in Figure 4.6. Giroud measured the extent of the matemal riboflavin depletion associated with various pregnancy outcomes which are summarized in Table 4.1. It is seen in Table 4.1 that riboflavin deficiency caused low birthweight, malformation or resorption of offspring at levels which caused no maternal signs.
TABLE 4.1
RIBOFLAVIN DEPLETION IN MATERNAL RAT LIVER FOR DIFFERENT PREGNANCY OUTCOMES: ALL ANALYSES AT 21 DAYS' GESTATION
| maternal liver riboflavin as | |
| percentage of normal 45 mcg/g |
| normally fed controls | 100 | ||
| outcome when deprived 4 or more days | |||
| before mating: | |||
| low birthweight pups | 80 |
| congenitally malformed pups | 65 |
| abortion or resorption | 54 |
| maternal signs of riboflavin deficiency | 50 | |
| maternal death | 35 | |
| Source: Giroud et al., 1949. |
Riboflavin and pantothenic acid are both coenzymes and animal reproduction has been reported to be upset if enzyme saturation falls below about 80 per cent (Esch et al., 1981). Levels of enzyme saturation appear to correspond approximately to the percentage liver contents in Table 4.1 and are in practice easier to measure. Eighty per cent instead of 100 per cent enzyme saturation would not be expected to produce signs of deficiency as tissues generally have enzyme reserves. Some explanation is needed of the susceptibility of the reproductive system to such a modest depression of enzyme saturation levels and to nutrient deficiencies that would not be noticed outside reproduction. The explanation is to be found in the susceptibility of the endocrine system to nutrient deficiencies discussed further below.
Later research by Potier de Courcy and Terroine (1968) also of the Paris Medical School confirmed Giroud's findings and referred to six other studies describing the modesty (legerete) of the deficiency needed to cause malformations. Pantothenic acid deficiency was also shown to produce malformed foetuses in 100 days without there being any "external signs" of deficiency. These experiments showed that rats and mice were more susceptible to damage from nutrient deficiency before and around the time of mating than at any other time in the life cycle. The same deficiencies later in pregnancy, or in young or adults, produced no apparent effects.
Giroud et al., (1950) also noted that the mother early in pregnancy had priority for riboflavin over the foetus. During the latter part of pregnancy, including human pregnancy, the placenta extracts vitamins from the maternal serum for the benefit of the foetus. The human baby has a higher blood serum riboflavin than his mother (van den Berg et al., 1978). The placental pump, so important in all the later stages of pregnancy, does not exist in the early stages which are therefore less protected. This was not understood by the early investigators, nor was it at first understood how the endocrine system enhances susceptibility to some nutrient deficiencies.
HORMONAL IMBALANCE CAUSED BY NUTRITIONAL DEFICIENCIES
Nelson et al. (1951, 1953) found that by injection of the ovarian hormones oestrone and progesterone they could restore the fertility of rat dams made infertile by pyridoxine deficiency before mating. They also found that injection of gonadotrophins (LH, FSH, prolactin) restored implantations to 78 per cent of controls but only 29 per cent had living young. Nelson (1953) concluded:
"Dysfunction of both the pituitary and the ovary is involved in the hormonal inadequacies of these vitamin-deficient animals."
Nelson and Evans (1955) found the effects of thiamin deficiency before mating illustrated in Figures 4.4 and 4.5 could be prevented and fertility could be restored by injection daily of lmcg oestrone and 4mg of progesterone.
It was concluded from these studies that the endocrine system mediates the effects of many nutrient deficiencies and inhibits reproduction before these deficiencies have any direct effect on germ cells or embryo. All nutrient deficiencies do not however act in this way. Thus the effects of folic acid or pantothenic acid deficiency on reproduction cannot be corrected by injection of hormones (Nelson et al., 1951). The secretion of many hormones is modulated by nutrition including for example growth hormone, somatomedins and insulin (Phillips & Vassilopoulou-Sellin, 1979). However the level of gonadotrophins appears to be affected at lesser degrees of nutrient deficiency than other hormones. In the course of evolution this characteristic probably had survival value preventing reproduction when food supplies were unsatisfactory. In the context of reproduction the endocrine system became the arbiter of nutritional adequacy. Ovulation ceased or implantation failed long before there was any real threat to the female. Luteinizing hormone (LH) appears to be depressed by an inadequate diet.
The effects of different nutrient deficiencies on hormonal balance need further study and are complicated by feed-back for example between ovarian hormones and the hypothalamus, which is part of the normal control of hormone levels. Riboflavin deficiency causes hormonal imbalance illustrated for oestradiol and progesterone in Figures 4.7 and 4.8 based on experiments on gilts by Esch et al. (1981) of the University of Illinois. Riboflavin is essential Figure 4.8.
Disturbance of progesterone levels in pigs. Source: Esch et al., 1981. Figure 3. for the homeostasis and liver clearance of these steroid hormones and a deficiency causes excessive accumulation which inhibits LHRH secretion by the hypothalamus and gonadotrophin secretion by the pituitary with resulting infertility. As discussed further below low maternal energy intake or protein intake also cause hormonal imbalance. During the early stages of reproduction including both ovulatory maturation and spermatogenesis a normal hormonal profile is a primary requirement of satisfactory reproduction. A defective diet is a diet that causes hormonal imbalance.
HORMONAL IMBALANCE CAN BE MUTAGENIC
In a symposium entitled "Mutations in Man" (Obe, 1984) Hansmann of the University of Gottingen suggested that failures of neuroendocrine control are a primary cause of chromosomal abnormalities and in particular of nondisjunction at meiosis I which happens in females during follicular development and concluded:
"Such alterations in normal follicular function may result from various failures at any level of the hypothalamic-pituitary-gonadal axis."
In particular reduced levels of gonadotrophins slow down follicular development, delay ovulation, and thereby increase the risk of error at meiosis I.
Individual hormones have been shown to be mutagenic in excess and also when deficient. Oestradiol, although an essential ovarian hormone, has been shown to be mutagenic in excess in mice and in human lymphocyte culture (Banduhn & Obe, 1985; Becker & Schdnreich, 1981). Progesterone in excess has been shown to be mutagenic in male and female animals including dogs, hamsters and rats. Excess progesterone interferes with meiosis in male and female animals and can cause a variety of trisomies and monosomies. The general effect of excess progesterone is to make animals infertile, but the animal experiments show that some cells with chromosomal abnormalities remain viable and proceed through meiosis although defective (Williams et al, 1971).
Lucille Hurley, of the University of California and one-time President of the Teratology Society, suggested that any factor that reduces embryonic DNA synthesis increases the risk of malformations (Eckhert & Hurley, 1977; Hurley, 1981). Most congenital malformations are associated with total numbers of cells and not reduced cell size. A slow-down in rates of cell replication caused by a slow-down in DNA synthesis increases the risk of disorganized differential growth and malformations. When the slow-down can be prevented by injection of ovarian hormones it is reasonable to conclude that it is a shortage of these hormones that is responsible for the slow-down, the growth disorganization and malformations. During early embryonic development immediately following fertilization progesterone is the hormone likely to be in short supply and to have these effects. Progesterone deficiency is a consequence of the corpus luteum having too few cells because of too low a rate of replication of the granulosa cells during follicular development. If follicular development proceeds too slowly there is a rise in mutations in surviving ova. Studies of the effect of maternal diets of different protein content show very clearly the increase in the proportion of defective ova produced before fertilization as the ovulation rate and rates of cell replication decline.
PROTEIN INTAKE AND THE FIRST DAYS AFTER FERTILIZATION
Animal experiments show that the hypothalamus is more sensitive to the protein content of diet than the ovaries, embryo or foetus. Nelson and Evans (1954) showed that a protein-free diet fed to rat darns from mating resulted in 90 to 100 per cent embryonic resorptions, the foetal deaths occurring in most cases in 9 or 10 days. Injection of oestrone (0.5ug) and progesterone (4mg) daily maintained pregnancy in 100 per cent of dams and the number of living young was close to normal, again suggesting that the effect of nutrient deficiences is via the hormone balance. The dams suffered major losses of body weight. Similar results were reported in experiments by Callard & Leathem (1970). Kinzey and Srebnik (1963) maintained pregnancy in rats fed a protein-free diet by using exclusively the pituitary hormones FSH, LH and prolactin. The availability of amino acids for follicular development and for embryonic and foetal growth is controlled by the endocrine system. Whether or not the protein in a particular diet is adequate for reproduction depends primarily upon the reaction of the hypothalamus to blood amino acid concentrations and not upon any direct effect of amino acids on the reproductive process, although individual amino acid deficiencies by themselves are mutagenic in cell culture (Freed & Schatz, 1969; Schemp......p & Krone, 1979).
Several studies have reported on the effects in animals of dietary protein restriction shortly before fertilization on embryonic cell replication immediately after mating. Mufioz and Malavd (1979) of the Venezuelan Institute of Scientific Research reported on these effects in mated female mice. It is seen in Table 4.2 that the mice on the lower protein diet produced fewer ova. The number of corpora lutea indicates the ovulation rate which is seen to be reduced by lowering the protein content of the diet. The ova were counted and few were lost before counting. It made no difference to the ovulation rate whether the rat dams were on the low protein diet for 2 weeks or 4 weeks before mating. This points to the existence of a period of heightened susceptibility to a low protein diet in mice lasting not more than 14 days.
TABLE 4.2
EFFECT ON PROTEIN INTAKE ON NUMBER OF OVA AND NUMBER OF CORPORA LUTEA IN MICE
| percentage protein | number of mice | average number | per dam of | |
| in diet | ||||
| ova | corpora lutea | |||
| 27 | 40 | 8.3 +/- 0.9 | 8.85 +/- 1.2 | |
| 8 | 40 | 5.45 +/- 1.0 | 5.6 +/- 1.5 |
Mufloz and Malavd examined the number of cell divisions in fertilized ova which happened 48, 72 and 96 hours after mating in rat dams which had been fed a standard or low protein diet during the 14 days before mating as shown in Figures 4.9, 4.10 and 4.11.
The low protein diet produced cells that replicated slowly in contrast to the standard diet which produced cells which replicated at the normal rate. There were, in effect, fast and slow replication lanes. The failure to proceed to first cleavage indicates a defective ovum and probably a defective hormonal profile during ovulatory maturation. A subsequent rate of cell division below normal indicates a slow-down in DNA synthesis and an increase in risk of low birthweight and malformation in survivors. It is seen in Figure 4.11 that 34 ova in the slow lane had not proceeded even to first cleavage at 96 hours after mating and were unfertilized or carried defects originating around meiosis I or II. At the same time 122 ova in the fast lane had divided to produce embryos with 32 or more cells, but none of the ova from the dams on the low protein diet had reached this stage. Assuming all the ova in the slow lane were not defective the slow-down must have been a consequence of depressed levels of progesterone and possibly of other hormones. The unsatisfactory hormonal profile of the mice on the low protein diet prejudiced normal development before mating during ovulatory maturation, after ovulation and during fertilization and subsequently during the early stages of embryonic development. The low protein diet reduced fertility by causing fewer ova to be ovulated, caused damaged ova to be produced that did not survive and, by slowing down cell replication during the early stages of embryonic development, reduced the viability of any embryos that did in fact survive.
Mice have a high rate of metabolism and low nutrient reserves. Pigs in contrast have a much lower rate of metabolism and high nutrient reserves. Pond et al. (1968) of Comell University showed that protein starvation of pigs instituted before breeding caused low or reduced fertility, low birthweight and stillbirth, in contrast to the slight effects of protein starvation during pregnancy. In pigs as in mice it is the protein status during the period immediately preceding mating that matters most.
The effects of a high as well as standard and low protein diet before mating on the reproduction of female mice have been reported by Tagami and Sudo (1982) working at the University of Ibaraki, Japan. The largest number of ova are seen in Figure 4.12 to have been produced by mice on a standard diet. Both low and high protein diets resulted in a lower ovulation rate. Mice are seen to be susceptible to percentages of protein in diet that are both too high and too low only one day before mating, a period which covers ovulatory maturation and meiosis I. Extension of the period on these diets before ovulation from 1 to 30 days had a further small but real effect on ovulation rate. It is shown in Figure 4.13 that the low and high protein diets also produced large numbers of abnormal ova. The classification of ova as abnormal by Tagami and Sudo would include the ova classified by Mufioz and Malav6 as failing to proceed to first cleavage or subsequent failure to continue dividing. Studies by Tagami and colleagues have also shown that both abnormally high and low protein diets increase the length of the reproductive cycle, increase the time from introduction of the male to fertilization, increase the number of pups dying during lactation and reduce the growth rates of surviving pups.
It may be asked why then is too much protein harmful? Protein has to be metabolized and this requires enzymes. A high protein diet can produce competition for a coenzyme between the enzymes needed to metabolize surplus protein and enzymes needed for other purposes including growth and reproduction. Three coenzymes which have been shown to be a limiting growth factor in this way are riboflavin, pyridoxine and biotin. The endocrine system is sensitive to the blood serum concentrations of all these three nutrients.
The reaction of the endocrine system to maternal body weight and to total food intake and the effect on reproduction are discussed further in the next chapter.
THE EFFECT OF NUTRITION ON SPERMATOGENESIS
The testes like the ovaries are dependent for their function on the secretion of reproductive hormones by the hypothalamus and pituitary. As in the female the secretion of these hormones notably FSH and LH is modulated by nutrition. It has been shown that B vitamin deficiency causes infertility and atrophy of the testes in male animals which can be corrected by administration of pituitary hormones or the testicular hormone testosterone (Mann & Lutwak-Mann, 1981). However it is difficult, as in the female, to separate the direct effects of nutrient deficiency on the male testicular function and the indirect effects mediated by the hypothalamus and pituitary. A study of underfeeding male rats concluded that "the basic problem in underfed rats is one of pituitary failure" (Howland, 1975). Deficiencies of vitamin A and zinc are examples of deficiencies that reduce the response of the cells of the testes to stimulation by testosterone. However vitamin A deficiency also disturbs the secretion of FSH by the pituitary (Huang et at., 1983), and the uptake of zinc by the prostate is dependent on pituitary hormones (Gunn & Gould, 1957; Millar et al., 1957).
When is spermatogenesis most susceptible to nutritional deficiency? Animals, usually mice, are used
extensively for testing chemicals for mutagenicity. Komatsu and a team from the University of Tokio
and the Japanese National Cancer Center noted that many chemicals under test reduced the food
intake. They therefore undertook an investigation using mice to see how far reduced food intake alone
was responsible for the mutagenicity rather than the chemical under test. A summary of some of their
results is shown in Figure 4.14 using morphological abnormality of sperm as the indicator. Komatsu
and his colleagues concluded that reduction of food intake is in itself mutagenic and that "chemicals that reduce food intake cannot be screened" using animals in this way. This team also answered the question about the comparative susceptibility of the different stages of spermatogenesis. They found that Type B spermatogonia and early spermatocytes were most susceptible to food restriction (Ko matsu et at., 1982). As might be expected from the discussion in Chapter Three illustrated in Figure 3.5 the highest susceptibility was during the time of maximum rates of DNA synthesis and cell replication.
Illness may cause men to go short of food for a week or a fortnight or longer. There is evidence that short term illness, such as appendicitis or tonsilitis, can cause important reductions in sperm concentration and motility, whether caused by the illness itself or short-term semi-starvation (David, 1982). A period of food restriction associated with illness is therefore a good reason for advising deferment of conception until Type B spermatogonia become available that were produced under conditions of health, that is 3 or 4 months from the retum to health.
Low mineral content of semen is associated with male infertility, abnormal sperm morphology and low sperm motility as illustrated in Table 4.3 based on research by Pandy et al. (1983), of the Health Division of the Bombay Atomic Research Center.
TABLE 4.3
ASSOCIATION OF SPERM ABNORMALITY AND LOW
SEMEN MINERAL CONTENT
| magnesium | calcium | zinc | |
| mg/dl | |||
| fertile men | 14 to 18 | 24 to 28 | 19 to 24 |
| 23 infertile men | 9.1 | 17.8 | 11.7 |
| 6 men with azoospermia | 5.7 | 16.0 | 8.1 |
| 9 infertile men with worst | 6.5 | 14.1 | 8.1 |
| sperm morphology | |||
| 8 infertile men with lowest | 6.3 | 14.9 | 7.5 |
| sperm motility | |||
Deficiencies of magnesium and zinc have been shown to be mutagenic in animals and to cause chromosomal abnormalities as illustrated in Figure 4.15 (Bell et al., 1975). Zinc deficiency has been shown to cause abnormal sperm morphology (Dinsdale & Williams, 1980). The association of infertility with low semen magnesium and zinc in Table 4.3 is not therefore unexpected, but cause and effect cannot be assumed because the men with low intakes of these particular minerals may have had low intakes of other nutrients. Low semen concentrations may also have causes other than low intake including malabsorption and high excretion. Low semen mineral content is nevertheless a useful indicator of possible impaired fertility and of possible defective diet. Takahara et al. (1982) reported reduced fertility in male patients at seminal zinc levels below I Smg/dl and found zinc supplementation effective in improving fertility in 50 per cent of cases. The expediency of zinc supplementation by Pandy's patients were deficient in magnesium not zinc and in others the effects of magnesium, zinc and calcium deficiency were probably additive.
The evidence is convincing that deficiencies of minerals other than magnesium and zinc can have serious effects on the fertility of farm animals. The correction of selenium deficiency is now established practice for animals grazing selenium deficient pastures. The most serious effect of selenium deficiency in domestic animals is on the fertility of the male (Wilkins & Kilgour, 1982). Bleau et al. (1984) of the University of Montreal have reported a significant correlation between sperm count and seminal selenium in man. Seminal selenium below 35ng/ml was found to be significantly associated with a low sperm count and a higher percentage of morphologically abnormal and non-viable sperm. The best pregnancy outcome in Bleau's series of 125 men consulting for infertility was associated with seminal selenium between 60 and 7Ong/ml and fertile controls averaged 67.4 b1 5.4ng/ml. High intakes of selenium are toxic and mutagenic. The US National Research Council recommends a daily adult selenium intake between 50 and 200mcg. The expediency of selenium supplements that ignore the magnesium, zinc and other minerals is however doubtful. If data on actual deficiencies are not available, and it is thought that supplementation is desirable, it is difficult to justify any but balanced supplements. However deficiencies are not confined to minerals.
The effects of vitamin A deficiency on the fertility of male animals has been a subject of research since 1933 (Mason, 1933). Vitamin A deficiency in animals causes morphological abnormality of sperm and if prolonged causes complete disintegration and disappearance of spermatids and spermatozoa and nearly all spermatocytes (Mitranond et al., 1979). Vitamin A is essential for DNA and RNA synthesis and for all cell replication (Omori & Chytil, 1982). Deficiency only appears to affect dividing spermatogonia, and the dormant, infrequently dividing, A0 spermatogonia appear in animal studies to be unaffected and able to regenerate cells, thus reviving spermatogenesis on restoration of vitamin A supply. Animal experiments throw doubts on the assumption that stores of vitamin A can be released quickly enough to protect germ cells if there is a sudden drop in intake. Some damage to sperm of male rats can be seen after exposure to a vitamin A deficient diet for only 72 hours in animals with adequate vitamin A stores (Kalla, 1981). Huang and Marshall (1983) stress that spermatogenesis in animals "is extremely sensitive to changes in serum vitamin A levels". It is nevertheless quite unclear whether or not the vitamin A deficiencies found in minorities of men in the western world are such as to interfere with spermatogenesis. The extensive research has been largely confined to animals and few research papers have been found that mention effects on men or women.
This chapter began with a discussion of the effects on reproduction, including spermatogenesis, of deficiencies of folic acid and cobalamin. Folic acid is one of the few nutrients for which there is both some information on the serum levels likely to affect spermatogenesis and on the prevalence of low serum levels in some populations. Thus a Canadian national survey found 11.2 per cent of Canadian men in the 20 to 39 age range to be at "high risk" with serum levels of folic acid below 2.5ng/ml which is inadequate to maintain the intracellular levels needed for efficient DNA synthesis (Canada, 1975).
In animal husbandry male animals used for breeding are valuable and their feeding receives great care because any degree of infertility is expensive. As a single bull may have 20,000 offspring fertility may be measured in births as a percentage of inseminations, for which his owner is paid. The fertility of different bulls is found to vary from under 20 per cent to over 80 per cent which is close to the upper limit imposed by average fertility of the cows. There is a substantial and growing veterinary literature on bulls, rams, goat-bucks; stallions, male mink, lions and other zoo animals showing that the right diet improves male reproductive performance and suggesting what is best. Mann and Lutwak-Mann (1981) in their treatise on "Male Reproductive Function and Semen" say:
"There is hardly any component of ordinary diet that could be pronounced as unimportant to male reproductive function..."
The attitude to the nutrition of valuable male animals used for breeding is to reduce risks to a minimum. This requires supplementation if there is any doubt about adequacy of the diet for such minerals as zinc, selenium, iodine or vitamin A. Excessive intakes of some essential nutrients including zinc, selenium, iodine, or vitamin A. Excessive intakes of some essential nutrients including zinc, selenium, iodine and vitamin A can increase mutation rate in animals and so increase risks and it is not therefore usual practice to supplement with more than reasonable physiological amouts of any nutrient.
Chapter Five includes a discussion of what are reasonable, desirable intakes of nutrients.