5 Chapter II: Low endogenous progesterone concentrations days
5.4.5 Relationships among cow-associated parameters
Older cows showed less problems during parturition (rs = - 0.214; P < 0.001), but more claw diseases (rs = 0.385; P < 0.001). The number of lactation affected MYAI positively (rs = 0.50; P < 0.001).
Chapter II: Discussion
5.5 Discussion
There is some evidence in literature [33, 59, 61] that high progesterone (P4) concen-trations during early pregnancy have a positive impactonembryonicdevelopmentand IFN-τ production.For thisreason, several studies on P4 supplementation have been conductedtoinvestigatethe possible beneficial impact of exogenous P4 on pregnancy rate(PR).However,resultsvaryconsiderably.Onereasonforthesedifferencescould be that only cows with a lack of endogenous P4 may benefit from an exogenous P4
administration. Therefore,themainobjective of this studywas toinvestigate whether there are differences in PR after P4 supplementation in cows with variously high P4
concentrations at the beginning of supplementation.
The level of P4 seems to be essential for pregnancy maintenance, as cows that be-came pregnant had significantly higher endogenous serum P4 concentrations on Day 5 than non-pregnant cows in the present study. This finding is in accordance with other studies in both beef heifers and dairy cows that detected positive correlations betweennaturallyoccurringhighconcentrationsofP4 in milk [46, 159, 160]andplasma [161, 170] in the immediate post conception period and the likelihood of conceptus survival and successful establishment of pregnancy. Recently, Kendall et al. [169]
reported an increased incidence of low P4 concentrations measured in the milk of repeat-breeder cows. The decisive influence of P4 on the outcome of insemination can be explainedthroughvariousmeansthatawardP4 afavorableimpact onembryo developmentandmaternalendometrium.Amongotherthings, P4 is known to suppress uterine myometrial activity [171-173], while inducing the production and release of manyuterine proteins[174, 175]andgrowthfactors [176, 177],as well as immunosup-pressive proteins [178], as soon as the embryo reaches the uterine body. Further-more, the uterine environment also affects the embryo itself. Recently, insulin-like-growth-factor-binding-protein-1, which is induced by P4, has been revealed as a com-monendometrialmarkerofconceptuselongationinsheepandcattle,whichmostlikely regulatesconceptuselongationbystimulatingmigration and attachment of the troph-ectoderm [48]. Besides the development of a suitable uterine environment, the establishment of a pregnancy in ruminants requires adequate conceptus elongation
Chapter II: Discussion
production. A positive relationship betweenhigh serum P4 during early pregnancy, conceptus elongation and the uterine concentration of the antiluteolytic factor IFN-τ has been demonstrated in cattle [59-61] and sheep [62]. As no investigations relating to embryo development, like embryo size and embryonic IFN-τ production, or relating to uterine environment, like measurements of uterine proteins and growth factors, have been carried out in our study, no reliable interpretation can be made in what way high endogenous P4 has mediated its positive impact on PR.
Progesterone supplementation with PRID Delta® induced a significantly sharper in-crease in peripheral P4 from Days 5 to 7 among P4-supplemented cows (PC) compa-red to control cows (CC) and therefore increased P4 levels significantly. This was in accordance with the results of previous studies that showed that the P4 increase from pre- to post-insertion samples was higher in supplemented cows [61, 179]orheifers [78]vs.controls.While, in the present study, the rise in P4 between Days 5 and 7 was about 2-fold higher in PC than in CC, Carter et al [33] noticed an approximately 3-fold increase in peripheral P4 throughout a similar interval (Day 3 to 5) induced by an exogenous P4 supplementation.
Progesteroneremainedsignificantlyelevatedin PC until Day 14, although profiles ten-dedto merge beginning on Day 7. A very early start of rapprochement of P4 values following the previous increase between PC and CC was also seen in another study in which P4 remained elevated in PC as opposed to CC for five days, after which temporary differences evened out [33]. There are various approaches to explain this observation: a considerably reduced drug release from the PRID over time, an en-forced metabolism of P4 in the liver or a repression of P4 production and release into circulation by the animal’s own CL due to the supplemented hormone.
Since PRID are produced to provide a continuous drug release for at least 7 days, and since it is proven that those devices are even able to provide a slow release of P4 up to 21 days [180], our first mentioned hypothesis of a reduced drug release of the PRID over time may probably not be true.
Through studies on rats [181] it could be ascertained that there are no short-term homeostatic mechanismsinvolvingthemetabolicclearancerate(MCR)of P4 thatcan
Chapter II: Discussion
respond to a change in P4 productionin ordertoreduce variationsinblood P4. How-ever, it seems much more the case that P4 homeostasis is limited to a long-term regulation of the MCR. In high-yielding dairy cows, an enforced MCR in terms of a higher rate ofglucuronidation and sulphation of P4 in the liver is mainly associated with rising milk yields and feed intake especially [14, 37]. Thereby, P4 inactivation in the liver is highly influenced by the diet which is fed and the source of energy that is used.AnincreasedMCRcanbeobserved with the usage of high-energy rations [182].
Since MCR of P4 is not submitted to any short-term homeostatic mechanisms and since all study cows were fed the same standard ratio and were evenly distributed on PC and CC by milk yield and lactation number, differences among cows regarding P4
cannot be explained by an enhanced MCR in the liver during the study period, either.
Thus, it could rather be assumed that P4 production by the CL was down regulated through the P4 supply.ThesynthesisandsecretionofP4 is mainly stimulated by luteini-zing hormone (LH) from the anterior pituitary, the main luteotropic hormone in cattle [183, 184], whose release is regulated by the hypothalamic gonadotropin releasing hormone(GnRH) [185, 186]. LH stimulates P4 production in small luteal cells via the LH receptor [187].Additionally, in luteal cells, it increases the expression of genes for the oxytocin (Day 6-10 of the estrus cycle) and progesterone receptor (Days 6-16 of the estrus cycle) [188] as well as of genes encoding for Steroidogenic Acute Regula-toryProtein(StAR), cytochrome P450 side chain cleavage (cytochrome P450scc) and 3β-hydroxysteroiddehydrogenase(3β-HSD)synthesis, which are required for steroid-genesis inthe mitochondria[189, 190]. ElevatedP4 concentrationsregulate themsel-ves by a negative feedback loop, controlling the hypothalamic–pituitary–gonadal axis andsuppressingtheGnRHrelease from the hypothalamus. As GnRH pulse frequency decreases, the frequency of episodic FSH and LH secretion also decreases. The CL consequently loses its FSH and LH receptors [185, 186]. Ireland at al. [191] noticed that in PRID supplemented heifers a significant increase of the amplitude of both LH and FSH episodes occurs after PRID removal. This negative feedback of PRID inser-tion on the hypothalamus and pituitary is used in the therapy of cystic ovaries. Re-ducing both LH concentration and pulse frequency, LH is insufficient for maintaining the cyst and it undergoes functional regression and atresia [192, 193]. This may help
Chapter II: Discussion
to explain why there was neither a positive nor a negative influence of PRID supple-mentation on pregnancy rate detectable in this study. Once a certain critical value has been exceeded, hormonal counter regulation may have started. Resulting from the reduced endogenous P4 synthesis, hormone levels between PC and CCevened out.Althoughthehypothalamic–pituitary–gonadalaxisis accepted as the most impor-tant regulator of luteal steroidogenesis, it must be born in mind that P4 homeostasis seemstobemorecomplex. Recently, it was described thattreatmentwithPRIDfor14 daysreduces subsequentCLvascularization,therebycausingadiminished P4 release into circulation on the following days [194]. As steroidogenesis and angiogenesisof the CL seem to be tightly related [90, 91], this might be an additional supervisory mechanismtocontrol peripheralP4.Furthermore,in bovineearlyCL, P4 productionis alsoefficientlyregulated by a variety of luteotropic factors like oxytocinandother pro-ducts oflutealorigin i.e. prostaglandins I2andE2, noradrenaline [184, 195].In addi-tion, P4 autoregulates and stimulates its own synthesis in the luteal cells of sheep, cows and rats via increased3β-HSDactivityorstimulatedgeneexpression for StAR protein and cytochrome P450scc on Days 6-16of the estrus cycle [196, 197]. More research must be carried out to fully understand this complex process.
In this study, endogenous P4 supplementation increased PR neither among cows with low nor among cows with intermediate or high endogenous P4 concentrations.
To this day, there is a controversial discussion about the benefit of P4 supplementa-tion on PR.While many studies have shown a benefit of this approach others have not and in many studies the number of cows was too small to allow worthwhile statistical analysis to be employed. However, it should be noticed that in general the number of animals used for the investigation was higher (n > 100) in those studies not suggesting an overall positive effect of exogenous P4 on PR [32, 77, 85, 88]
compared to those that did [32-34, 84]. Futhermore,it is conspicuous that in several studies a numerical difference (~ 5%) [85, 88, 198] or even a tendency [199, 200] but no significance for higher PR in P4-supplemented compared with control cows could be observed. This may indicate that there are many external factors influencing the potential positive impact of an exogenous P4 supplementation on PR. For instance, Stevenson et al. [200] revealed a tendency for interactions of conception rates after
Chapter II: Discussion
P4 supplementation with the studied herd. Moreover, ameta-analysis [201] of 17 P4
supplementation studies emphasized the importance of the onset of supplementation to its benefit on PR. It was observed that overall supplementations started before Day 6 improved PR by 10% while supplementa-tions started thereafter did not. The importance of an early and sufficient rise of P4 was further strengthend by a finding of Lamming et al. [202], which were able to show that a delay in P4 rise is associated with a progressive shortening of the luteal phase which itself is accompanied by a progressive reduction in PR. But also a too early postovulatory P4 rise directly after AI (Day 0-2) seems to exert a negative influence on PR. This could be due to a has-tened transport of the embryo from the uterine tube into the insufficiently prepared uterus body [203-206] or to the simulation of an advanced stateofcycleby thePRID therebyinducingluteolysisbefore the embryo is mature enough to produce sufficient quantities of IFN-τ t. Furthermore, the duration [84] and the amount of supplemen-tation [9] as well as the fertility status of the supplemented herd seem to be decisive for the efficiency of a P4 supplementation after AI. With regard to the last aspect, according to Mann et al. [201], most improvementcouldbeseenif initially herd fertility was low (< 50%). This was not confirmed in our investigation.Eventhoughfertilityof bothherdswasinitiallylow(PRafterfirstAI< 40%), no improvement in PR through the use of PRID was recognizable. Last of all, a recently published study by Forro et al.
[199] has shown that in case of an Ovsynch application the combination with a P4
supplementationhas a positive effect on PR. Consequently, also the usage of certain hormones and synchronization programs for estrus induction may have some influence on the efficiency of an exogenous P4 supply.
In the current investigation, cows with a natural heat had significantly higher P4
concentrations on Days 19 and 21 compared to cows with a PGF2α-induced heat.
This may be due to the fact that PGF2α administration had a slightly negative effect on pregnancy rates in this study, with the result that cows with natural heat became pregnant about 10% more likely. This is in clear contradiction to most investigations on that topic so far, which could not detect any considerable difference in the PR between cows with PGF2α-induced and natural heat [207-209]. In some cases
[210-Chapter II: Discussion
as a reproductive management tool to improve PR. However, consideration must be given to the fact that in these investigations the positive effect of PGF2αwas primarily attributed to a marked improvement of estrus detection. Due to the experimental set up, this aspect did not play any role in our investigation. It is a well known fact that a sufficiently strong and long P4 exposure in the estrus cycle preceding ovulation is needed for an adequate lutealfunction in the subsequent cycle, which in turn ensures embryo survival. In several investigations [213-215], it was found that administering consecutive injections of PGF2α early in the estrus cycle (Days 3 and 4) reduces the capacity of the CL to secrete P4 within the same cycle. So far little is known about the effect of estrus induction with PGF2α on P4 concentration, P4 receptor expression and uterine environment in the subsequent cycle. However, the stage of the estrus cycle at which synchronization is initiated seems to influence the reproductive success of a timed artificial insemination protocol [216]. This could be confirmed by Schönkypl et al. [217], who were able to demonstrate that estrus induction with PGF2α may negatively affect fertility in cattle as a premature regression of the CL is followed by a reduced uterine P4 receptor expression and a modified pattern of uterine secretory functions. This might explain why pregnancy rates between cows with PGF2α-induced and natural heat differed significantly, although both groups exhibited similar P4
concentrations in early and mean luteal phase in this study.
Chapter II: Conclusion
5.6 Conclusion
Overall, our results emphasize that P4 plays a decisive role in bovine reproduction and that the amount of P4 after AI is of vital importance for the successful establish-ment of a pregnancy.However, the expected improvement of fertility by a supple-mentation with P4 for 14 days did not materialize in this study, even though intra-vaginal PRID significantly raised systemic P4 in circulation for about 10 days. It seems that thefertilitystatusoftheherd,theamount of medication, the point of com mencement and the duration of supplementation are crucial for the potential benefit of an exogenous P4 supply. An excessive duration of supplementation may have been the reason for the lack of benefit from P4 supplementation detected in this study.It is thereforeproposed to specifytheoptimumpointin timefor PRID removal through a higher frequency of blood sampling in order to prevent the repression of endogenousP4 production due to MCR. Another possibility to avoid MCR could be to reduce medication to an amount that prevents the activation of the negative feedback loop while, on the other hand, it approves P4 to stimulate its own syntheses via the 3 β–HSD pathway. Moreover, further investigations on the exact influence of P4 on oocyte quality, preparation of the endometrium, and development of the early embryo will provide valuable information for possible interactions with P4 supplementation.
Chapter II: Figures and Tables
5.7 Figures and Tables
Figure 5: Treatment schedule of postpartum dairy cows randomly treated with a pro-gesterone releasing intravaginal device (PRID Delta®) from Days 5 to 19. Estrus of all cows was synchronized with prostaglandin F2α (PGF2α). Cows were artificial insemi-nated(AI)onDay 0 (Ovulation = Day 1). Blood samples (BS) were taken on all days shown. Ultrasonographic pregnancy diagnosis was performed on Day 30.
Figure 6: Changes of serum P4 concentration between Days 5 and 21 after insemination in cows with (supplemented) and without (control) P4 supplementation via PRID Delta® (Ceva, Libourne, France). ⃰ Differences between P4-supplemented (PC) and control cows (CC) (P < 0.05) on the days are indicated.
Chapter II: Figures and Tables
02468
5 7 14 19 21
LPC LPP IPC IPP HPC HPP
Days
progesterone in ng/ml
Figure 7: Changes of serumP4 concentrationbetween Days 5 and 21 after inseminationin P4-supplemented cows with low (LPP), intermediate (IPP) or high (HPP) endogenous P4 on Day 5 and their associated control (LPC, IPC or HPC).
Chapter II: Figures and Tables
Table 3.1: Progesterone(P4)concentrationsofP4-supplemented(PC)vs.control cows (CC) and, pregnant (P) vs. non-pregnant (NP) cows and all cows (Total) at different days of observation. Data are means, standard error of mean (SEM), number of cows (n) and minimum (Min) and maximum (Max) values.
P4 Day5 P4 Day 7 P4 Day 14 P4 Day 19 P4 Day21 CC Mean + SEM 1.41* + 0.04 2.88* + 0.10 6.01* + 0.15 5.82* + 0.21 4.83* + 0.22
n 235 131 196 210 192
Min –Max 0.1 -4.5 0.1 – 7.2 0.1 -17.5 0.1 -19.3 0.1 -13.6 PC Mean + SEM 1.46* + 0.04 4.75# + 0.12 6.57# + 0.16 5.91* + 0.21 4.67* + 0.24
n 192 144 166 190 167
Min –Max 0.3 -3.5 1.5 -12.3 0.6 -14.7 0.5 -16.0 0.0 – 11.8 Total Mean + SEM 1.43 + 0.03 3.86 + 0.10 6.27 + 0.11 5.86 + 0.15 4.76 + 0.16
n 427 275 362 400 359
Min –Max 0.1 -4.5 0.1 -12.3 0.1 – 17.5 0.1 -19.3 0.0 – 13.6 NP Mean + SEM 1.36* + 0.04 3.72* + 0.13 6.10* + 0.15 5.10*+ 0.19 3.57* + 0.21
n 256 156 217 245 220
Min –Max 0.1 -4.2 0.1 -12.3 0.1 -17.5 0.1 -16.0 0.0 -17.7 P Mean + SEM 1.54# + 0.05 4.02* + 0.14 6.54* + 0.15 7.08# + 0.19 6.65# + 0.16
n 166 117 143 154 138
Min –Max 0.4 -4.5 1.5 -7.3 2.6 -12.3 1.0 -19.3 0.2 – 13.6 Total Mean + SEM 1.43 + 0.03 3.85 + 0.10 6.28 + 0.11 5.86 + 0.15 4.76 + 0.16
n 422 273 360 399 358
Min – Max 0.1 - 4.5 0.1 – 12.3 0.1 -17.5 0.1 – 19.3 0.0 – 13.6 Values with different superscripts (*#) within columns differ (P < 0.05).
Chapter II: Figures and Tables
Table 3.2: Progesterone(P4) concentrations of P4-supplemented cows with low (LPP), intermediate (IPP) or high (HPP) endogenous P4 on Day 5 and their associated control (LPC, IPC or HPC) on different days of estrus cycle.
Data are means, standard error of mean (SEM) and number of cows (n; in brackets).
supplementation
ABCD Values with different superscripts within columns differ (P < 0.05).
‡^#•
Values with different superscripts within the same row differ (P < 0.05).
Chapter II: Figures and Tables
Table 3.3: Relative proportion (%) and total number (n) of pregnant cows among groups of P4-supplemented cows with low (LPP), intermediate (IPP) or high (HPP) endogenous P4 on Day 5 and their associated control (LPC, IPC or HPC)
Group according
to serum P4 Day 5 LPC LPP IPC IPP HPC HPP
Pregnancy rate in
% [n]
25.8 [17]
26.7 [12]
42.0 [42]
42.7 [38]
43.1 [28]
50.9 [29]
Übergreifende Diskussion