The major objectives of trial 1 were to ascertain whether CB154 treatment inhibited PRL release in local ewes and whether this delayed lactogenesis. Secondary objectives were to determine the period of CB154 treatment necessary to delay lactogenesis and to identify any evidence for the existence of "type I" and "type II" ewes as described by Kann et al. (1978).
Treatment with CB154 in trial 1 was successful in reducing PRL to very low levels and in reducing early lactation milk yields. The lack of an effect of CB154 on progesterone concentrations, and the lack of a consistent effect on insulin concentrations, implies that differences in milk yields between the groups can probably be attributed to changes in circulating PRL levels. The fact that CB154 treatment has been found previously to have no effect on plasma concentrations of progesterone in ewes (Niswender 1974), or progesterone, placental lactogen, oestrone sulphate (Forsyth et al. 1985), GH, insulin or thyroxine (Hart 1976; Hart and Morant 1980) in goats, lends further support to this conclusion. However, reports that CB154 increased GH (Forsyth et al. 1985) and decreased insulin (Hart and Morant 1980; Johnsson et al.
1986) concentrations cannot be ignored, and the possibility remains that CB154 exerts its effects through some factor other than PRL. The situation with respect to insulin is confusing, since Hart has reported apparently conflicting results (Hart 1976; Hart and Morant 1980) relating to the same trial. The first report (Hart 1976) indicated no consistent effects of CB154 on insulin concentrations in goats, whereas the second report (Hart and Morant 1980) showed that long- term CB154 treatment (23 or 77 d, concomitant with oestradiol benzoate and progesterone treatment to induce lactation) reduced insulin concentrations during a subsequent 17-d milking period. This result is contrary to the finding in trial 1, in which CB154 treatment for 20 d (and perhaps also for 9 d) increased plasma insulin concentrations. No logical relationship is apparent between the higher prepartum insulin concentration in CB20 ewes and subsequent depressed milk yields.
As noted earlier (section 3.2), the statistical test for a delay in lactogenesis was the interaction CB20 versus E/S by time (i.e. testing the hypothesis that CB154 treatment depressed milk yields in early lactation but that CB154-treated ewes eventually attained the same milk yields as E/S ewes). In trial 1 this interaction was not significant, but there was a significant main effect of CB154 treatment. Moreover, visual examination of the data (Figure 3.4) suggests that milk yields of the 3 treatment groups were converging towards the end of the milking period. Thus it can be concluded that CB154 treatment was effective in delaying lactogenesis (i.e. in delaying the onset of normal milk secretion). Since the two CB154-treated groups did not differ in milk yield (averaged across ranks) it is apparent that the 9 and 20 d periods of CB154 treatment were equally effective in depressing milk yields.
CB154 treatment was, however, more effective in reducing milk yield in twin-bearing than in single-bearing ewes when used for the longer 20 d period (Figure 3.17). For example, the ratio of milk yield on day 1 of lactation for the E/S : CB9 : CB20 groups was 1 : 0.46 : 0.68 for single- bearing ewes whereas it was 1 : 0.46 : 0.11 for twin- bearing ewes. In fact, on the first day of lactation milk production was completely inhibited in two of the twin-bearing ewes, and almost completely inhibited in another two, but only completely inhibited in one single-bearing ewe. The 9 d CB154 treatment period reduced milk yields by about the same proportion in single- and twin- bearing ewes. In contrast, accumulation of precolostrum in the udder was not affected by long- term bromocriptine treatment in does carrying twin foetuses but in single-bearing does it was delayed about 4-6 weeks (Forsyth et al. 1985).
The mechanism by which long periods (20d) of CB154 treatment differentially affected milk yields in the single- and twin-bearing ewes is unclear. The interaction between effects of length of CB154 treatment (9 versus 20 d) and rank (single- versus twin-bearing) on plasma PRL levels was nonsignificant, indicating that different lengths of CB154 treatment had the same effects on plasma PRL concentrations in single- and twin-bearing ewes. Thus the differential effects on milk yield cannot be explained by corresponding effects on plasma PRL concentrations during the pre- or postpartum periods. This leaves two other possible explanations. Circulating PRL during the period 20 to 10 d prepartum may have an important effect on milk yield in twin- but not single- bearing ewes with the result that suppression of PRL levels during this period reduces milk yields in the former group but not in the latter group. Alternatively, the effect of the CB154 may be mediated via other hormones (but apparently not progesterone or insulin). For example, twin- bearing ewes have higher plasma concentrations of oPL than single-bearing ewes at this time (Gluckman et al. 1979; Oddy and Jenkin 1981) and CB154 treatment might have affected these levels. However, Martal and Lacroix (1978) and Forsyth et al. (1985) could find no effect of CB154 treatment on circulating PL concentrations in ewes and does respectively, although Martal and Lacroix (1978) reported that CB154 treatment increased placental oPL concentrations 4 to 6 times.
The lack of differences between the treatment groups in lactose percentage over the first 7 d of lactation indicates that lactose synthesis was inhibited by the treatments to the same extent as milk volume. Indeed, since lactose synthesis is the major factor determining milk yield (Linzell and Peaker 1971b) it is assumed that the CB154 treatment delayed lactogenesis by delaying the initiation of normal lactose synthesis. There is no apparent explanation for the difference in lactose percentage between the CB20 and CB9 groups over the weekly intervals.
Interpretation of other milk composition results was complicated by the differences in milk yields. The lack of differences in milk fat percentage suggests that milk fat synthesis was inhibited to the same degree as milk volume (i.e. lactose synthesis). The much higher protein content measured in the secretion collected during the first few days of lactation from the CB154-treated groups was
probably due initially to immunoglobulin secretion not being inhibited relative to milk volume. This is entirely possible since the immunoglobulins in colostrum are transferred from the blood rather than being synthesised in the glandular epithelium. The secretion collected from CB154-treated ewes had a thicker consistency than that from E/S ewes on the first day or two, and in ewes producing very low volumes it was often so thick as to make removal very difficult. However, since the milk composition analyses measured only total protein it is not possible to determine changes in glandular protein synthesis during the initial stage of lactation. Similar results have been reported in cows; prepartum CB154 treatment depressed the onset of lactation, reduced the concentration of lactose, increased protein concentration and altered the casein composition of colostrum (Karg and Schams 1974). Johke and Hodate (1983) reported that CB154 injections during the 2 weeks prepartum markedly decreased concentrations of α-lactalbumin and lactose in the colostrum but that ß-lactoglobulin and IgG concentration increased. Since large concentrations of immunoglobulins are normally present in milk only during the initial stage of lactation, when the pool of immunoglobulin which has accumulated during the prepartum period is washed out (Newstead 1976), the continued difference in protein percentage past the first few days in the present trial cannot readily be attributed to differences in immunoglobulin secretion. The immunoglobulin transfer from the blood is probably unaffected by CB154 while the synthesis of lactose, and hence milk volume, is delayed. This would result in an increase in the concentration of immunoglobulin in milk which, because of the increased viscosity and reduced milk volume, might take longer to be washed from the gland. This may only partly explain the increase in milk protein concentration since its continued elevation for 4 weeks indicates that protein synthesis was less affected by CB154 treatment than was milk volume (i.e. lactose synthesis). These results suggest that initiation of normal milk protein synthesis is not controlled by PRL alone.
Examination of the plasma PRL profiles of individual ewes for the presence of "type I" and "type II" ewes was necessarily limited to the E/S group, since CB154 treatment suppressed PRL concentrations in the other two groups. There was no suggestion, within this limited sample (n=9), of the existence of the two "types" of ewe. The progesterone profiles of all ewes were examined, since CB154 had no effect on mean progesterone concentrations, and these also provided no evidence of the existence of "type I" and "type II" ewes. However this trial was not specifically designed to identify such differences and no statistical test was considered appropriate.
Having determined that CB154 treatment for 9 d or more prepartum effectively reduced plasma PRL concentrations and delayed lactogenesis, it was necessary to establish that the responses to CB154 were effected by changes in circulating PRL concentrations. This was tested in trial 2, by comparing the effects of oPRL with those of bicarbonate in CB154-treated ewes, and with the effects of ethanol/saline treatment. Based on the results of trial 1, it was intended to administer
CB154 for 9 d prepartum, but ewes lambed on average 2 d earlier than expected, resulting in a mean prepartum CB154 treatment period of 7 days.
In trial 2, completed in autumn, plasma PRL concentrations were much lower than those in trial 1 despite the fact that ewes in both trials were exposed to the same photoperiod during the experimental period. Longer photoperiod increases PRL levels greatly in wethers (Trenkle 1978; Eisemann et al. 1984a and 1984b) and in both pregnant and lactating ewes (Munro et al. 1980; Perier et al. 1986). It is likely that the pattern of change in photoperiod prior to each trial had a carry-over effect on PRL levels. In fact, recent evidence indicates that circannual rhythms are generated by an endogenous process that is synchronised by exposure to long days during the previous spring and summer (Karsch and Wayne 1988; Malpaux et al. 1989; Wayne et al. 1990). It is also possible, although unlikely, that the different composition of the diet in the two trials (spring pasture versus autumn pasture) was responsible for the differences in circulating PRL levels. These possibilities will be addressed in Chapter 5.
Plasma PRL levels were lower in the E/S than in the CB154-treated ewes before treatment began (Figure 3.18) which can only have been due to a chance effect associated with the random assignment of ewes to treatment groups. This difference was essentially maintained from the start of CB154 treatment until parturition, suggesting that CB154 had no effect on plasma PRL concentrations. It is possible that this dose of CB154 was unable to decrease PRL concentrations further than the naturally low levels recorded in the autumn. Although blood sampling did not coincide with the mean date of parturition, the periparturient peak in plasma PRL concentrations was evident in three E/S ewes and it is therefore probable that all E/S ewes had such a peak whereas CB+BIC ewes did not. The oPRL administration would have restored plasma PRL levels in CB+PRL ewes to values similar to the normal peak, as indicated by the data presented in Figure 3.1.
The administration of oPRL (CB+PRL group) resulted in milk yields similar to those in the E/S ewes and significantly greater than those in the CB+BIC ewes, indicating that oPRL prevented the CB154-induced reduction of milk yields. Administration of PRL in CB154-treated cows also resulted in restoration of milk yields (Akers, Bauman, Capuco et al. 1981).
Milk yields in CB+BIC twin-bearing ewes were substantially reduced by CB154 treatment (e.g. to 38% of E/S group yields on d1), but the reduction was much greater in single-bearing ewes (e.g. to 5% of E/S group yields on d1). This difference must be interpreted with caution because there were only 2 ewes in the CB+BIC group and, perhaps as a consequence, the CB+BIC versus E/S by rank interaction was nonsignificant with respect to milk yield. However, plasma PRL concentrations were reduced by CB154 treatment to a greater extent in single- than in twin- bearing ewes during the period d 2 to d 7 postpartum (Figure 3.25). This may explain the low milk yields of single-bearing CB+BIC ewes during early lactation. However it does not explain the
inconsistency in effects of 9 d (trial 1) versus 7 d (trial 2) CB154 treatment on milk yields of single-bearing ewes. These results are likely to be confounded by the seasonal differences between trials 1 and 2. It was noted that milk yields were higher in the spring-lambing ewes than in the autumn-lambing ewes (2082 ±133 versus 1732 ± 92 g/d in the E/S ewes).
Treatment with CB154 had the same effect on milk composition in trial 2 as it did in trial 1. Milk fat and lactose content were unaffected while protein concentration was increased. Administration of oPRL prevented the CB154-induced increase in milk protein concentration, presumably by increasing milk yield (lactose synthesis) by a greater proportion than it did protein synthesis.
To summarise, these trials have shown that CB154 treatment for 7 d or more prepartum was effective in delaying or inhibiting complete initiation of lactogenesis. Since the injection of 0.5 mg/kg LWT oPRL resulted in plasma concentrations similar to those observed during the normal periparturient surge, and reversed the effects of CB154, it was concluded that PRL has an important role in enabling the rapid and complete initiation of lactogenesis. Varying responses to the different lengths of CB154 treatment, especially between single- and twin-bearing ewes, suggested that the timing of oPRL administration may affect subsequent lactation, but the success of the 2 daily oPRL injections indicated that this frequency and the doses used were adequate for subsequent trials. It was considered necessary to further investigate the relationship between the seasonal differences in PRL levels and milk yields, and to determine whether nutritional factors contributed to these differences. Furthermore, it was considered appropriate to investigate the effectiveness of alternative routes of oPRL administration, especially direct administration into the udder. This might confirm a direct action of oPRL on the mammary gland (without the need for an intermediate hormone) and could lead to an effective method of increasing milk yields by oPRL supplementation.