S. Landau et al. Animal Reproduction Science 64 2000 181–197 187
experiments 1, 2, 3 and glucose experiments 2, 3 were tested by analysis of variances using a General Linear Model GLM of the Statistical Analysis Systems Institute 1988.
Analysis for experiment 2 was according to a Latin-Square design including dietary treat- ment, cow and feeding cycle as main effects. As plasma glucose and insulin may be
affected by DMI Richards et al., 1989, which is itself related with days post-partum and milk production Hart et al., 1978, variancecovariance analyses were carried out on mean
plasma insulin and glucose values, using DMI, milk production and days post-partum as covariates.
A repeated measure procedure with cow treatment as the term of error in the GLM was used in experiment 3 to analyse dietary effects on circulating insulin and glucose. The
status preovulatory versus subordinate of individual follicles was established, relying on estradiol concentration in FF, the status being a main effect in the GLM. In order to assess
the effects of diet, preovulatory follicular status and their interaction on insulin and glucose, the statistical significance of differences between group means was evaluated using Tukey’s
studentised range tests.
The effects of diet and preovulatory follicular status on insulin and glucose were re- evaluated in cows from which FF had been aspirated from pairs of one preovulatory and
one subordinate follicle by using paired T-test.
3. Results
3.1. Experiment 1: circulating insulin during the luteal and the follicular phases Average values for plasma insulin did not differ significantly between the luteal phase
0.349, S.E.M. 0.03 ngml and the follicular phase 0.417, S.E.M. 0.07 ngml phases of the oestrus cycle. However, variation in insulin concentration during the follicular phase was
twice greater than in the luteal phase. The highest value for insulin was recorded at 16±10 h before the occurrence of standing oestrus behaviour. When the average concentration of
insulin during the luteal phase is considered as baseline value 0.349 ± 0.030 ngml, the highest measured insulin concentration in the follicular phase 0.712 ± 0.170 ngml was
approximately twice the baseline value. This peak above baseline was significant P 0.03 in cows that conceived, and tended P 0.10 to be significant in cows that did
not conceive. Therefore, in subsequent experiments, in order to prevent confounding the effects of nutrition and of day-in-cycle, nutritional effects were measured in the luteal phase
following a synchronised oestrus.
3.2. Experiment 2: short-term dietary effect on plasma insulin during the luteal phase Daily DM intake 21.8 ± 1.4 kg and milk yield 34.0 ± 2.5 kg were not affected by
dietary treatment, but the cow effect was significant on both P 0.03 and P 0.0001, re- spectively. Diets consumed were reasonably similar to diets planned +0.5, +0.5, −0.5 kg
DM for control, SBM and CGM treatments, respectively. The effects of cow and of sampling time were significant P 0.001 but these factors
did not interact with dietary treatment effects on insulin. No significant peak in insulin
188 S. Landau et al. Animal Reproduction Science 64 2000 181–197
Fig. 1. a Plasma insulin on X-axis — hours relative to diet distribution, Y-axis — insulin ngml and b glucose on X-axis — hours relative to diet distribution, Y-axis — glucose gl concentration in six cows fed alternatively
a standard total mixed ration control, △, or the same diet supplemented with corn gluten meal CGM,
j
or soyabean meal SBM,
r
: means ± S.E.
concentration was noted, although more elevated insulin concentration was noted at 12 h after meal distribution.
Dietary treatments tended P 0.06 to affect the mean plasma concentration of insulin Fig. 1a, which averaged 0.696±0.054, 0.625±0.054 and 0.504±0.054 ngml for the SBM,
CGM and control diets, respectively. Plasma insulin was affected by days post-partum P 0.01, but not by milk yield. When DM intake was used as covariant, insulin concentrations
averaged 0.679 ± 0.050, 0.650 ± 0.054 and 0.479 ± 0.054 ngml for the SBM, CGM and control diets, respectively, and the statistical significance of dietary effect on insulin was
increased. Feeding the SBM and CGM diets was associated with 29.5 and 26.4 higher mean plasma insulin compared with the control P 0.05 and P 0.01, respectively.
When days post-partum was used as covariant, insulin concentrations were highest for SBM 0.741±0.058 ngml, and lowest for control 0.479±0.054 ngml, P 0.02, CGM being
intermediate 0.650 ± 0.054 ngml.
Milk yield or days post-partum had no effect on plasma glucose. The plasma concentration of glucose Fig. 1b was not affected by dietary treatments but a significant cow effect
was recorded P 0.0002. There was a significant decline in glucose from the time of distribution of the ration P 0.02 but this did not interact with dietary treatment.
S. Landau et al. Animal Reproduction Science 64 2000 181–197 189
3.3. Experiment 3: short-term dietary effects during the luteal phase on insulin and glucose in the follicular fluid
The daily intake of DM 24.0 ± 1.2 kg and daily milk yield 38.9 ± 2.1 kg did not differ between nutrition groups. Intake of DM tended to be greater in April than in January
P 0.07, especially in cows fed CGM P 0.02. Overall, plasma insulin concentration was 21 higher after 7 days of feeding the exper-
imental diets than in the pre-experimental period, when all cows were fed the basic total mixed ration Fig. 2, 0.733 versus 0.579 ngml, respectively, P 0.05. This was particu-
larly notable at 4 and 6 h P 0.05 after meal distribution. However, experimental dietary treatments did not differ in mean plasma insulin. Plasma glucose did not differ between the
experimental diets and the basic ration.
If all diets were combined, preovulatory follicles had higher insulin and glucose concen- tration in FF than subordinate follicles 0.282 versus 0.127 ngml, P 0.0001, for insulin;
0.614 versus 0.386 gl, P 0.002, for glucose; Table 2. The insulin concentration in the FF of cows fed the CG diet was 26 higher than in their counterparts fed CGM P 0.04,
Fig. 2. Plasma insulin on X-axis — hours relative to diet distribution, Y-axis — insulin ngml concentration in cows fed total mixed rations before open symbols and after full symbols 7 day inclusion of: a ground corn
grain
s
and
d
, n = 5; b corn gluten meal
h
and
j
, n = 6 or c soyabean meal
e
and
r
, n =
5: least square means ± S.E.
190 S. Landau et al. Animal Reproduction Science 64 2000 181–197
S. Landau et al. Animal Reproduction Science 64 2000 181–197 191
SBM being intermediate. When statistical analyses of insulin concentration was run sepa- rately for preovulatory and subordinate follicles, dietary effects did not reach significance
in subordinate follicles, whereas preovulatory follicles ranked CG CGM P 0.03, SBM being intermediate Table 2.
As expected, the volume of FF was greater for preovulatory than for subordinate follicles 1.68 versus 1.26 ml, P 0.04.
The content of insulin in each follicle was significantly affected by follicular status. Pre- ovulatory and subordinate follicles contained 0.490 and 0.150 ng of insulin, respectively
P 0.0001. The total intrafollicular insulin was also affected by diet P 0.02, but not by the diet × follicular status interaction. Insulin content in FF from CG-fed an-
imals was more than twice that of the CGM-fed cows 0.44 versus 0.21 ng, P 0.002, and 32 higher in CG-fed cows than in their SBM-fed counterparts 0.44 versus 0.31 ng,
P
0.05. The total intrafollicular content of glucose was also significantly affected by follicu-
lar status. Preovulatory and subordinate follicles contained 1.02 and 0.450 mg of glucose, respectively P 0.0001. The total intrafollicular content of glucose was significantly af-
fected by diet P 0.006, but not by the diet × follicular status interaction. Intrafollicular content of glucose was 46 higher in CG-fed than in CGM-fed cows 0.99 versus 0.53 mg,
P
0.002 and 32 greater in CG-fed cows than in their SBM-fed counterparts 0.99 versus 0.54 mg, P 0.02. When statistical analyses of insulin and glucose intrafollicu-
Fig. 3. Relationship between intrafollicular insulin on X-axis ngml and glucose on Y-axis gl in cows fed total mixed rations supplemented with ground corn grain
d
, n = 5, corn gluten meal
j
, n = 6 or soyabean meal
r
, n = 5.
192 S. Landau et al. Animal Reproduction Science 64 2000 181–197
lar content were run separately for preovulatory and subordinate follicles, dietary effects reached significance in preovulatory, but not in subordinate follicles Table 2.
Only four cows had simultaneously a preovulatory and subordinate follicle. In these cows, FF from preovulatory follicles had higher concentration of insulin than subordinates
P 0.02. Diet P 0.03 and follicular status P 0.007 affected insulin total intrafollicular content P 0.03. Ranking of diets for total intrafollicular insulin was not
modified CG SBM CGM.
The relationship between insulin and glucose in FF is shown in Fig. 3. A linear correlation coefficient of 0.66 P 0.001 was found between them. However, it seems that glucose
concentration plateau at 0.6 gl when insulin was greater than 0.167 ngml. When calculated separately for both classes of follicles, the correlation was significant for preovulatory
R = 0.75, P 0.005, but not subordinate follicles. Significant correlations were also found between oestradiol and insulin R = 0.73; P 0.0001 and between oestradiol
and glucose R = 0.52, P 0.001. However, significance disappeared when correlations were calculated separately for the two classes of follicles.
4. Discussion
