Discussion

A central cavity was observed within 69.4% of the investigated CL, which is in accor-dance with results of Kastelic et al. [133], who detected a cavity within 79% of the CL.

However, acavity was found more often than in most previous examinations so far [134, 138, 139]. Approximately 75% of the cavities inthepresentstudyweresmaller than 1.0 cm² in size. Cavities >5 mm in diameter can exclusively be observed in a developing CL [134].Therefore,itcanbeinferred that a cavity was detectable within the majority of CL for the following reasons: firstly, no cut-off value for the presence of a cavity was set in this study, in contrast to previous investigations [133, 140];

secondly, only developing CL with a higher incidence of cavities within the CL [134]

were evaluated; and finally an advancedultrasoundmachineofhigh-qualityand high-resolution was used, which might have been able to make smaller cavities visible.

Both a big cavity within the CL as well as much luteal tissue caused CL with great CS and DCL. It fact, it can be said, that the greatest CL, where those, that exhibited both.

As shown in previous investigations [26, 133, 134, 138] and as confirmed by the current investigation, the existence and size of a luteal cavity neither correlated with LS nor with LBF or P4. In contrast, Grygar et al. [141] reported thatcowswith luteal cavities displayed higher plasma P4 levels. Ultimately, pregnancy rate was neither affected by cavity presence nor by cavity size in this study, which is in agreement with previous investigations [138, 140].

The mean cross sectional areas reflecting CS and LS as well as the mean P4 con-centration on Day 5 p.i. found in this study are fairly in agreement with those reported in previous studies [113, 136, 137].Minordifferences can beexplained by the various number of sampled animals, the use of a different method of analysis (RIA instead of EIA)aswellasadifferent testing media (serum instead of plasma).Therewasa mode-rate but highly significant correlation between all parameters of corpus luteum size (PCS: DCL, CS, LS) and P4 detectable in the present study. Smaller corpora lutea were associated with alower P4 concentration on Day 5. This largely correspondsto prevalent assessments in literature, which attribute P4 secretion and concentration a

Chapter I: Discussion

the diameter of CL (DCL) [18, 25, 120], in particular in the first days of cycle, the phase of CL growth and development [142]. While in most investigations the detec-ted correlation was rather moderate [111, 112] as was the case in the current study, there has been a study [120] describing a somewhat higher correlationbetweenDCL and plasma P4 concentration. The positive relationship between PCS and P4 has also been verified in studies using milk as the medium for the detection of P4 concen-tration [21, 25, 143]. Also CL weight on Day 5 p.i. is known to correlate positively with systemic plasma P4 concentration [27, 28, 142].Since LS represents the entire hor-mone producing tissue, it could be concluded that LS might exhibit a stronger co-herence with P4 than the other PCS. However,itappeared that LS showed no greater consistence withP4 concentrations than CS and DCL in this investigation. An expla-nation for this could be due to a different capability of P4 synthesis of luteal cells having the same size. In an in vitro study [144], a reduced response to exogenous LH and PGE2has been shown in luteal cells from subfertile cows leading to a decrea-sed production of P4. It was concluded that luteal inadequacy, due to a diminished response to circulating luteotrophic hormones, may contribute to embryo mortality in subfertile cows.

With the exception of a very weak association of luteal blood flow (LBF) with CS and LS, there was no significant relationship of any PLBF with PCS or P4 in this study. This is in clear contrast to previous findings on that topic, which reported that LBF gra-dually increases in parallel with the increase in corpus luteum volume and plasmaP4

concentrationin the early CL [136, 137]. While Acosta et al. [111] assessed changes of LBF, LS and P4 only in terms of time, without evaluating the validity of LBF measurementstopredictP4 at acertainpointintime,Herzogetal. [137], who conduc-ted LBF measurements during one entire estrus cycle, additionally emphasized LBF to be superior as an indicator for luteal function rather than LS especially during luteal regression phase. In most phases of the cycle, their overall correlation between LBF and P4 was high (r = 71; P<0.001). Sinceanactiveangiogenesis and adequate blood supply is highly relevant to enable a physiological luteal development and to empower the CL to produce and release P4 in the circulation [74, 111], it is not surprising that numerous studies with both humans [103, 145, 146] and animals

Chapter I: Discussion

[91, 147, 148] have shown that P4 is clearly related to an enhanced blood supply and it is altogether difficult to understand why no conclusion with respect to thefunctional statusoftheCLcouldbedrawnbymeasuringLBFinthisstudy.Nevertheless,there are different explanatory approaches for this difference. Comparing our results with those of Herzog et al. on Day 5, it is noteworthy that LBF was more than one third higher in their investigations compared with our results, while LS was even slightly higher in our investigation. During angiogenesis of the growing CL more and more superficial arterioles branch out into centralizing arterioles of the parenchyma, which then dis-solve into a network of small capillaries [99]. The difference in LBF between those two investigations may be attributed to the ultrasonic devices and transducers utilized. Our ultrasound examinations were performed under field conditions in two dairy farms with a portable Doppler ultrasonic device equipped with a 7.5 MHz linear-array transducer, whereas Herzog et al. used a stationary ultrasonic device equipped with a 10 MHz linear-array transducer for their examinations. This stationary device may have provided a higher resolution capacity for slower LBF velocities within vessels of the smaller sizes. Another reason for the inconsistency in LBF may be that blood sampling and Doppler US measurements were conducted only at one definite time in cycle shortly after AI and, therefore, no day-by-time interrelation but exclusively suitability for diagnosis of the functional status of the CL at a certain point in time could be estimated in our investigation. Systemic P4 is known to vary slightly over the day, due to a pulsatil release from the bovine CL during early luteal phase [149]. The same applies to blood flow through arteries. [150]. This circadian rhythm of the artery blood flow may be attributable to temporarily effective, vasoactive peptides whose existence has been confirmed in the CL [151, 152]. While these changes in blood flow of the uterine and ovarian artery correlate significantly with changes in P4 concentration in humans [153] and cows [154] throughout the main parts of the cycle, they seem to be independent from the daily hormonal fluctuations [150, 155] during the periovulatory period.Thus far, it is not known exactly whether the same applies as with blood flowin theovarianstroma,follicularwallorCL.Since ranges of P4 and LBF were still narrow at this stage of the cycle, it can be assumed that one single measurement of P4 and LBF might not have allowed a reliable

Chapter I: Discussion

assessment of luteal function shortly after AI. In addition, Bourne et al. [156] stated that in the first days of the menstrual cycle blood vessels of a human CL are always maximally dilatated and LBF is exclusively influenced by blood pressure. This is because the blood vessels of the early CL neither possess smooth muscle cells nor are supplied with a vegetative innervation. If this should apply also for cattle, it would explain why there were strong individual differences.

Further studies should be conducted to investigate to what extent P4 and LBF show a circadian rhythm in cattle. Possibly, for scientific purposes,a more elaborate experi-mental protocol, including a combination ofmultiple measurements on a given day, may be a suitable means to ensure clearer discernibility of CL with different functio-nal status by colour Doppler sonography.

Although no significant linear relationship between P4 and pregnancy outcome was observable, there were considerable numerical differences in pregnancy outcome between cows withvariousP4 concentrations (low/ intermediate/ high) on Day 5 p.i..

The higher the P4 the higher was the risk of pregnancy. This is in agreement with results from previous investigations in humans [92, 157, 158] and cattle [46, 159-161], which have shown that there is a clear link between the endogenous P4 con-centration and the likelihood of pregnancy in the immediate post insemination period.

The number of cows used in this study was lower than in previous investigations.

Possibly, it might have been too small to achieve statistical significance.

In a recently published study [162], a positive correlation between pregnancy status and the dimensions of the CL was proven on Days 10, 20, and 25 after AI in Italian Mediterranean buffalo cows.Pregnant cows also showed a greater rate of CL growth between Days 5 and 10 after AI.Due to the marked coherency of P4 with PCS proven in this study, it can be assumed that a causal connection also exists between the likelihood of pregnancy and PCS on Day 5. These hypotheses should be confirmed in a study on a larger number of cows.

With theexception ofa reducedLBFinnon-inseminatedcows(P<0.05),no relation-shipbetweenPLBF and pregnancyoutcomecouldbedetected in the present study. To the best of our knowledge, a significantly lower level of LBF in non-inseminated

com-Chapter I: Discussion

paredtoinseminatedcowshas never been reported before. Yet, upon closer exami-nation of a graphic from a previous study [113; Fig. 1 (A)], it becomes evident that LBFof non-inseminated cows was at least numerically reduced compared to LBF of all inseminated cows on Day 6.In both investigations the number of animals in the non-inseminated group was relatively low (n < 15).However, given that the signify-cance, measured in thisstudy, was extremely high it wouldbeinterestingto determine whethertheresultisrepeatableon alargernumberof cows. If this is indeed the case, it would be important to investigate by means of which mechanisms fertilized and un-fertilized ova exhibit various influences on LBF this early in the cycle.

The ascertained lack of relationship between LBF and pregnancy outcome is in con-trasttomanystudiesinhumans,whichwereabletoverify an interrelation between LBF and likelihood of pregnancy on several occasions [92, 157, 158]. Within cattle, Utt et al. [114] were the first to evaluate the suitability of color Doppler US measurements of the corpus luteum surrounding the time of natural luteolysis as a direct indicator of an early pregnancy. According to their results, LBF proved to be a useful indicator of CL status (differentiation between functional or regressing). Moreover, they were able to detect differences in LBF between cows with various pregnancy outcomes on Days 17, 19 and 21. However, since not only pregnant cows but also cows with EEL showed high LBF values on Days 17, 19 and 21, color Doppler ultrasound was no reliable method for a positive early pregnancy diagnosis. It is noticeable that a diffe-rent phase of the cycle was investigated in this study, and the results are therefore hardly comparable with our results. Comparable results with those of Utt et al. [114]

regarding the reliability of color Doppler ultrasound as a diagnostic tool for pregnancy diagnosis during the late phase of the estrus cycle were achieved by Herzog et al.

[113] in a recent report. The last cited authors additionally investigated LBF in earlier phases of the estrus cycle, pointing out no differences in LBF between pregnant and non-pregnant cows shortly after AI, which is consistent with our findings. Conse-quently, we conclude that discernible differences in LBF between pregnant and non-pregnantcowsexistnot earlier than Day 15 p.i.,atwhich a critical phase for the estab-lishment of a pregnancy begins [163]. Differences in LBF in the late phases of the cycle are not difficult to explain since it is known that the luteolytic cascade is

trig-Chapter I: Discussion

gered by severalvasocontrictive substances [164] and a reduction of LBF is a main component of PGF2α-induced luteal regression [111, 165]. At the end of diestrus, changes in LBF complywithmorphologicalchangesintheCL,likeregressionof capil-laries and degeneration of luteal parenchymal cells. According to the best of our knowledge, up to now, there are no PCS cut-offs in literature in order to differentiate cows with functional (P4 > 1.0 ng/ml) and subfunctional (P4 <1.0 ng/ml)CL in early luteal phase. Sofar, thefocus ofcut-off determination lay on identifyingcows with a PGF2α-responsive CL at different stages of cycle. Therefore, a comparison of our results with those findings established in previous investigations is possible only to a limited extent. Nevertheless, the determined P4 threshold value of 1.0ng/mllargely correspondswithP4 cut-offsselectedinpreviousstudies[118-120, 166].Cows with a P4 below cut-off hada smaller(2.3vs.2.5cm)DCL than cows with a P4 abovecut-off, which was in conformity with former findings of Bicalho et al. [119]. The same coherency applied for CS (3.4 vs. 4.1 cm²; P = 0.001) andLS (3.0 vs. 3.6 cm²; P<

0.001). The PCS cut off values, determined for later stages in the cycle [119, 137], were considerably higher than those we have established for Day 5. In accordance with previous investigations[119, 137],contradictorytrendsof SNandSPinthecase of increasing cut-off values had a negative effect on measurement reliability. There-fore,SNwasunsatisfactorilylowwithaSP of>90%.Moreover, it has to be noted that, due to the experimental protocol, a temporal inaccuracy of + 6 h of the time of ovula-tion has to be taken into account in the present study. Individual differences in P4 are still low in the early phase of the cycle. Therefore a tiny temporal inaccuracy with regard to the time of ovulation may have already had a significant impact on the amount of the measured P4 and on the reliability of the ascertained cut-off. Further-more,P4 release is subjected to a circadian pulsatile release into circulation. These inaccuraciescan only be compensated by a larger number of different measurements to confirm the time of ovulation and determine P4. Thus, it seems to be impossible to utilize the correlation between P4 and Pcs for a reliable evaluation of the functional status of a CL within a reasonable expenditure of time.

Chapter I: Conclusion