Results

4.4.1 Clinical findings

Single ovulations were detected in 128 cows (114 inseminated and 14 non-insemi-nated), whereas 10 cows without signs of estrus, 7 cows without contemporary ovu-lation and 5 cows with double ovuovu-lations were not compliant to study requirements and had to be excluded from further examinations. The cows used were (mean + SEM) 3.2 + 0.1 years (min-max: 2.0 – 7.0 years) old and in their 2.1 + 0.2 lactation (min-max: 1.0 – 5.0). At the time of first AI they were 66.7 + 1.3 days in lactation.

Their milk yield at first AI was 38.1 + 0.8 kg/day (min-max: 18.0 – 61.7 kg / day) per cow. Their average BCS was 3.00 + 0.04 (min-max: 2.00 – 4.00); and their average LNS was 1.7 + 0.1 (min-max: 1.0 – 4.0). A CL with cavity was found in 69.8% (n=90) of the cows (76.7% P [n=33], 64.5% NP [n=20], 65.0% with EEL [n=26] and 64.4% NI [n=9]).

4.4.2 Pregnancy rates and resulting assignment to groups

Forty of the inseminated cows were considered as non-pregnant to the AI of the previous cycle and inseminated again. Twenty-nine (72.5%) of those cows showed signs of estrus and exhibited milk P4 values < 3 ng/ml until Day 25, whereas 7 cows (17.5%) were identified to be in heat only by a milk P4 < 3 ng/ml and 4 cows (10%) showed signs of estrus, while milk P4 was still above 3 ng/ml. On Day 30 pregnancy diagnosis was performed on the remaining 74 cows without estrus until Day 25.

During the initial pregnancy diagnosis (Day 30) pregnancy outcome of three cows was uncertain. Within the repeated examination three days later two cows proved to be pregnant whereas in one cow no embryo was found. Ultimately, 43 inseminated cows (37.7%) were diagnosed as pregnant within the first gestation examination, whereas in 31 cows (27.2%) early embryonic loss had occurred. Fourteen cows were not inseminated.

With the exception of two cows (4.7%), which aborted between first (Day 30) and second (Day 42) pregnancy diagnosis, and one cow (2.4%), in which fetal loss

Chapter I: Results

4.4.3 Descriptive statistics of P4 concentrations, PCS and PLBF

Relative changes of progesterone concentration during cycle as well as means, ma-ximum and minimum values, SD, SEM, quartiles and ranges of parameters of corpus luteum size (PCS) and luteal blood flow (PLBF) at Day 5 p.i. are presented in Table 2.1.

4.4.4 Correlations among reproductive and cow-associated parameters and with P4, PCS and PLBF

The start of cycle after parturition (SCP) tended to occur later in older cows with more lactations (rs = -0.29, P = 0.055) than in younger ones. SCP showed a low negative relationshiptoCS(rs =-0.29)aswelltoLS(rs = -0.29) and accordingly LBF (rs = - 0.33;

P < 0.05 each).In addition CS (rs = 0.34, P < 0.001) and LS (rs = 0.27, P < 0.05) were positively related to the size of the preovulatory follicle (SPF) to minor degrees. Older cows had larger SPF than younger cows (rs = 0.24, P < 0.05). The remaining reproductive and cow-associated parameters had no influence (P > 0.05) on PCS and PLBF. Similarly, there were no effects (P > 0.05) of reproductive and cow-associated parameters on P4 concentrations with the exception of LNS, age of the cow and lactation number. Older cows (rs = - 0.20) with more lactations (rs = - 0.19) showed slightly lower levels of P4 than younger ones (P < 0.05). Furthermore, on most days of P4 measurement, P4 was reduced in cows with claw diseases indicated by a correlation coefficient of rs = - 0.29 (Day 5; P < 0.01), rs= - 0.26 (Day 9; P < 0.05) and rs = - 0.32 (Day 14; P < 0.05), respectively. LNS itself was positively related to the age of the cow (r = 0.35, P<0.01). The age of the cow also influenced calving ease (rs = - 0.30, P < 0.01) as well as milk yield at AI (rs = 0.60, P< 0.01).

4.4.5 Relationships among P4 concentration, luteal size and luteal blood flow

The intra-class correlation coefficients for the intra-observer reproducibility of CS, LS and LBF measurements were 0.98, 0.98 and 0.96, respectively.

There were extremely significant (P < 0.001 each) positive relationships among most parameters of corpus luteum size (PCS) like CS and LS (rs = 0.81), CS and CVS (rs = 0.58), CS and DCL (rs = 0.85), LS and DCL (rs = 0.74) as well as between CVS and

Chapter I: Results

DCL (rs = 0.46). The only exception to this was the relationship between LS and CVS, which were not related to each other (rs = 0.12, P < 0.001).

Similarly, parameters of luteal blood flow (PLBF) were extremely correlated (P < 0.001;

each) with each other (LBF vs. rBF: rs = 0.87; LBF vs. aBF: rs = 0.90; rBF vs. aBF:

rs = 0.97)

To assess possible relationships of PCS or PLBF with P4 and in order to validate pre-dictability of P4 by those parameters, scatterplots were charted (Figure 2) and corre-lations determined. The same parameters showing a weak coherency to P4 in the scatterplots exhibited moderate positive correlations with serum P4 concentration on Day 5 with regard tocorrelation coefficients:CS(rs =0.42), LS (rs = 0.41) and DCL (rs

= 0.41, P < 0.001 each). In contrast, CVS as well as PLBF were not related (P > 0.05) with the P4 concentration. Among PCS and PLBF, LBF showed very weak coherencies with CS (rs = 0.22, P < 0.05) and LS (rs = 0.30, P < 0.001).

4.4.6 Relationships of P4, PCS and PLBF with pregnancy outcome

There was no relationship (P > 0.05) between any of the PCS (CS, LS, CVS and DCL) and pregnancy outcome (pregnant, EEL, non-pregnant, not inseminated).

In contrast, there was an association (P < 0.01) between PLBF and pregnancy status.

LBF, rBF and aBF were greater in pregnant, EEL and non-pregnant cows than in cows that were not inseminated (P < 0.05; Table 2.2), yet there were no differences in PLBF among inseminated cows (P > 0.05).

Not until the end of estrus cycle (Days 19 and 20 p.i.) were differences in P4 concen-tration ascertainable between various pregnancy groups. From this point on, P4

decreased within NP (P < 0.01), while it remained on a higher level within P and EEL (P > 0.05; Figure 3). However, by allocating the cows into groups of different P4

levels on Day 5 (low, intermediate, high), a numerical difference in pregnancy outcome among cows with various P4 levels during early luteal phase has been proven. Pregnancy outcome was numerically, but not significantly higher in groups with higher P4 concentrations (low: 33.3%, intermediate: 37.3% and high: 47.6%

pregnant cows).

Chapter I: Results

Moreover, pregnancy status was only dependent on QE (P = 0.0304) as well as age (P = 0.0280) and LAC (P = 0.0352) of the cow.

4.4.7 Evaluation of cut-offs

To compare the usefulness of PCS and PLBF for assessing luteal function and in order to distinguish reliably between cows with reduced and high serum P4 concentrations, cut-off values should have been determined initially for each of the parameters mentioned above. However, due to the lack of coherence between PLBF and P4 and due to a very high individual variability of PLBF (min-max CV: 40.0 – 58.1%), derivation of cut-offs was conducted considering only DCL, CS and LS. These three parameters similarly exhibited relatively strong individual differences (min-max CV:

16.6 - 30.7%). The means and ranges were 2.39 and 1.6-3.4 cm for DCL, 3.83 and 1.8–6.8 cm² for CS and 3.37 and 1.7–5.7 cm² for LS, respectively. At Day 5 p.i., 30.0% of the cows had a P4 concentration < 1.0 ng/ml. Cows with a P4 concentration

< 1.0 ng/ml had a smaller (P = 0.016) mean CL diameter than cows with P4

> 1.0 ng/ml at Day 5 after AI (2.3 vs. 2.5 cm). The same applied for CS (3.4 vs. 4.1 cm²; P = 0.001) and LS (3.0 vs. 3.6 cm²; P < 0.001). First, a logistic regression analysis was used to determine the cut-off values for CS, LS and DCL at which, with a probability of more than 50%, P4 was > 1.0 ng/ml. These cut-offs were 1.97 cm for DCL, 2.46 cm² for CS, and 2.51 cm² for LS. In subsequent calculations, cut-off values with an optimized relation of SN and SP were calculated. Here, it was noticeable that overall SN declined and SP increased as cut-offs for DCL, CS and LS increased (Table 2.3a-c). The DCL cut-off value at which SN and SP were optimized for detection of a functional CL (P4 > 1.0 ng/ml) was > 2.11 cm (Figure 4a).

Optimized cut-offs for CS (Figure 4b) and LS (Figure 4c) with the least likelihood of error were > 3.13 or > 3.25 cm², respectively.

Chapter I: Discussion