PROGESTERONE CONCENTRATIONS IN MILK OF SUPEROVULATED COWS

Kudlac E., J. Konecny, J. Cermak: Progesterone Concentrations in Milk of Superovulated Cows. Acta vet. Bmo, 60,1991: 171-179. Ovarian response of donor cows after the administration of PMSG was lower (0-24, average 4.36 ± 5.65 embryos) than after the administration of FSH (0-41, average 9.27 ± 11.82 embryos). Better results of the superovulation were observed . in donor cows with high levels of milk progesterone (> 5.0 ng/ml) on the first day of the superovulatory stimulation and on the day of PGF2alpha administration than in cows with lower values or subsequent slow and insufficient drop in progesterone concentrations. Correlations were established between the numbers of corpora lutea, the number of embryos recovered and the level of progesterone in milk of donor cows superovulated by PMSG from day 4 after insemination and in the case of superovulation by FSH already from day 2 after insemination. Milk progesterone concentration assays are useful to provide a substantial and fairly objective information on the level of the superovulatory response. Cattle, superovulation, FSH, PMSG, ovarian response, progesterone' The transfer of embryos is becoming an integral part of a complex system of controlled reproduction in cattle. The method can be used to accelerate the breeding progress significantly and its application can be recommended in ·spite of considerable technical and financial problems involved. Procedures for practically all the necessary steps have been established and mastered, and generally good results are achieved in many countries (Hahn 1985, 1989; Greve 1986, inter alia), including Czechoslovakia (Holy 1988). • In the complex of problems related to embryo transfer, the decisive role is played by the effectivity and reliability of the superovulatory response and thus the number of transferable embryos is crucial. It should be noted, however, that in spite of an enormous effort. and extensive research work, the question of superovulation remains mostly unanswered and no examination procedure has been established that would allow an objective evaluation of suitable donor cows, which would guarantee a stable level of ovarian response and a constant recovery of a sufficient number of embryos. It is a well-known fact that the level of superovulatory response and its final results characterized by the number of good-quality transferable embryos depend on the breed, age, nutrition and health, the conditions at the parturition, puerperium, sexual cycle and the immediate condition of the ovaries, the method of superovulation and the gonadotropin used, the method of embryo recovery, and also other factors (Greve 1982; Kudlac and Holy 1984; Majerciak 1989, etc.). Among the limiting factors for an accurate assessment of suitable donor cows and successful superovulation Holy (1988) includes a balanced metabolism, a regular pattern of the reproductive cycle, including fully functional corpora lutea, and a sufficient sensitivity of donor cows to the administered hormone. A weaker response and frequently missing ovulation, together with low levels of progesterone in cows with metabolic disorders, particularly acidosis and ketosis, have already been demonstrated by our group (Vinkleret al. 1983; Kudlac et ale 1985). Most recently, we have been

The transfer of embryos is becoming an integral part of a complex system of controlled reproduction in cattle.The method can be used to accelerate the breeding progress significantly and its application can be recommended in •spite of considerable technical and financial problems involved.Procedures for practically all the necessary steps have been established and mastered, and generally good results are achieved in many countries (Hahn 1985(Hahn , 1989;;Greve 1986, inter alia), including Czechoslovakia (Holy 1988).

•
In the complex of problems related to embryo transfer, the decisive role is played by the effectivity and reliability of the superovulatory response and thus the number of transferable embryos is crucial.It should be noted, however, that in spite of an enormous effort.and extensive research work, the question of superovulation remains mostly unanswered and no examination procedure has been established that would allow an objective evaluation of suitable donor cows, which would guarantee a stable level of ovarian response and a constant recovery of a sufficient number of embryos.
It is a well-known fact that the level of superovulatory response and its final results characterized by the number of good-quality transferable embryos depend on the breed, age, nutrition and health, the conditions at the parturition, puerperium, sexual cycle and the immediate condition of the ovaries, the method of superovulation and the gonadotropin used, the method of embryo recovery, and also other factors (Greve 1982;Kudlac and Holy 1984;Majerciak 1989, etc.).
Among the limiting factors for an accurate assessment of suitable donor cows and successful superovulation Holy (1988) includes a balanced metabolism, a regular pattern of the reproductive cycle, including fully functional corpora lutea, and a sufficient sensitivity of donor cows to the administered hormone.
A weaker response and frequently missing ovulation, together with low levels of progesterone in cows with metabolic disorders, particularly acidosis and ketosis, have already been demonstrated by our group (Vinkleret al. 1983;Kudlac et ale 1985).Most recently, we have been  donors with lower response able to confirm (Vinkler et al. 1990;Kudlac 1990) more frequent variations of ovulation and lower levels of progesterone in cows subjeci:ed t~ an acidotic condition when superovulated by PMSG.
The purpose of our work was to study the development of progesterone concentrations in donor cows before and after superovulation and to determine the relation between the progesterone concentrations and the results of superovulation.

Materials and Methods
The concentrations of progesterone were studied in two groups of cows b#ore and after the su~ perovulatory treatment.The aim was to ascertain to what extent this method provides objective criteria for the selection of donor cows and checking of the superovulatory response.
In the first group consisting of 45 cows, progesterone levels were estimated by RIA in fat-free milk (Hruska et aI., 1978).FSH (Folicotropin inj.Spofa) and PMSG (Bioveta Ivanovice na Hane) were used as stimulants, in some cases anti-PMSG serum (Antisergon, Bioveta Ivanovice na Hane) was also used.In ,the second group (54 animals) progesterone was determined by RIA in full milk (Pichova .eta!. 1977) and superovulation Wlj.S initiated by FSH (Folicotropin inj.Spofa) donors with 4-12 CL donors with 1-3 CL only; The administration of hormones in combination with PGF2 alpha was carried out in accordance with standard methods -qsedin Czechoslovakia (Kudlac and Holy 1984), uswilly after once or twice induced luteolysis.The relation between the progesterone concentratioIis on tl;J.e.sepa.;.rate days before and partiCularly after the superovWatory treatment and the results of the superovulation was statistidilly, evaluated using the sequence .correlation by Spearman (Reisenauer 1970). .

Results and Discussion
While the differences in progesterone levels in individual cows prior to super-: ovulatory treatment were relatively very large, the results nevertheless provided a good'information on the character of the cycle and particularly on the activity of corpora lutea.The response to the superovulatory treatment was more constant in cows where progesterone levels corresponded to a normal oesttous pattern and a fully functional corpus luteum.However, no significant relation was demonstrated between the progesterone concentrations prior to or at the beginning of the superovulatory treatment and the quantitative or qualitative features of the superovulatory response (the number of ovulated follicles and developing corpora lutea), although certain trends were clearly discernible (Figures 1 and 2).Generally speaking, the results of the superovulation assessed according to the number of corpora lutea-and the embryos recovered varied greatly both Progesterone level assay was found to be a highly objective method of monitoring luteolysis after PGF2alpha administration (Figures 1 and 2).
A clearly significant relation (Tables 1 to 4) was ascertained in both groups of cows between progesterone levels and the superovulatory response (the number of corpora lutea or the number of embryos recovered).In donor cows treated with PMSG, the relation was found significant on the day after insemination (P < 0.05) and highly significant on day 6 (P < 0.01).When FSH was administered, the relation was found significant on day 2 after insemination and highly significant on day 3 to 4 (P < 0.01).
In the second group of cows, milk progesterone concentrations before and after • superovulation were assessed with regard to the success of superovulation.Progesterone concentrations of 5.0 ng or more in 1 ml milk in the luteal phase with fully functional corpora lutea were considered normal, while lower concentrations were regarded as deviations.At the beginning of the luteal phase (day 4 after oestrus) and on the day of oestrus, concentrations of 3 ng/ml or more and 0-2 ng/ml progesterone respectively, and at least 20 ng/ml in cows with a good superovulatory response were considered normal.The correlation between the percentage of cows with progesterone level deviations and the success rate of superovulatory treatment is shown in Tab. 5. Cows with the least response which were not even flushed; showed highest deviations in progesterone concentrations on nearly all days of the assessment, while the percentage of deviations in cows with a good ovarian response and the largest yield of viable embryos was minimal (Tab.6).
An individal evaluation of the relation between progesterone levels at and after the beginning of the superovulatory treatment and its results showed that the most +++ standard results were found in the donor cow No. 16105 (Fig. 3).This is a very good dairy cow with a regular sexual cycle" stabilized function of the corpus luteum and progesterone production" moreover with a regularly occurring onset of new fully functional cycle after each flushing and administration of PGF2a1pha.Much better ovulation rate and more constant character of ovarian response were found after the administration' of FSH than after PMSG.The average number of embryos recovered from 22 cows in the first group treated with PMSG was 4.36 ± 5.65 (min.0 and max.24) and 9.22 ± 11.82 (min.0 and max.41) from 23 cows treated with FSH.On average 6.2 embryos were recovered per donor in the second group.
Our investigations support the opinion that pituitary FSH can produce more constant results in superovulation than the administration of PMSG.The highly standard character of the superovulatory response of some donors is probably determined by a large number of internal and external factors" mainly the firm constitution and a high stability of the neuroendocrine system.The final superovulatory effect achieved in our study is comparable to the data by other authors  both in Czechoslovakia and in other countries (Monniaux et aI. 1983;Hahn 1985;Greve 1986;Baumung 1986;Krontodd et aI. 1986;Holy 1985Holy ,1987;;Maj,erciak 1989, inter alia).
A critical review of our results and the data puplished by many other authors (Kitzig et..aI. 1984;Kweon ~t aI. 1986;Yadav et aI. 1986;Goto et aI. 1987, inter alia) suggest 'that the assessment of milk .progesterone concentrations in cows before and after superovulation is very effective and can serve as an important seC;Qn4ary criterion in the final selection of donor cows for the stimulation anq.as a valuable source of infor,mation for the assessment of the ovarian response to superoVulatory treatment performed.We confirmed the data by Elsaesser (1981), Greve (1986),Callessen et aI. (1986), Stubbings and Walton (1986), Yadavet aI.( 1986) that low levels of milk progesterone in cows on the first day of a superovulatolj treatment indicate poor results of superovulation even though the presence of the corpus luteum 9n the ovary may be clearly demonstrated.

Table l '
EvaluadoD of the Reladon between ProlresteroDe Levels aud the NUlDber of CorpO!l'8 l u .by MeaDS; of Sequcage CQrreladoD Test after SpearllUUl (Group.I) EvaluadoD of the ReladoD between Progesterone Levels aud the Number of Embryos Rec:overed by MeaDS of Sequcage CorreladoD Test after Spearman (Group 1)

Table 5 Relation
Between Resuti. of SuperovulatiOD in Cows and Prosesterone LeVd on the Day of FSH Administration (total n = 38.Group 2) Number of Corpora I Number of Embryos I Numbers of Transfer- "