THE THERAPY OF OVARIAN CYSTS IN CATTLE , AND THE CHANGES IN BLOOD COUNT DURING ITS COURSE

KudIac E., K. Turecek, J. Vesely, and M. A. Siddiqui: The, Therapy of Ovarian Cysts in Cattle and the Changes in Blood Count during its Course. Acta vet. Bmo, 62, 1993: 79-88. Synthetic hypothalamic preparations of GnRH, produced locally, were administered in the cows of Czech Red and White breed and its crosses with Black and White' cattle. There was no difference in the results after the use of DIRIGESTRAN inj. Spofa (5 ml and 10 mI, i. e. 200 pg and 400 pg of gonadorelin, respectively)and SUPERGESTRAN inj. Spofa (4 mI, i. e. 100 pg of the analogue with prolonged and stronger biological activity). The results were significantly worse with the use' of lower dose of SUPERGESTRAN inj. Spofa (2 mI, i. e. 50 (.tg). Altogether 23.9% (26.9%) a smaller number of cows were treated successfully (P < 0.01). The average age of the whole group before the treatment was 4.2 years. The average age of the successfully and unsuccessfully treated cows was 3.63 years and 5.50 years, respectively (P < 0.01). No statistically significant differences were foimd in the red blood counts before and after the treatment. Concerning the white blood count, a significanf' difference was found between the relative proportion of neutrophil granulocytes with segmented and non-segmented nucleus before and after the treatment (P < 0.05). A highly significant decrease (P < 0.01) in both absolute and relative numbers of lymphocytes occurred after the treatment. The successfully treated animals showed also a signifiCant decrease of the relative percentage of monocytes (P < 0.05). ' Cattle, syndrome of ovarian cysts, GnRH, red blood count, white blood count The problem of ovarian cyst syndrome in cattle 'is being solved in veterinary medicine for~ decades. Therapy has been the main point for the research, with respect to the current knowledgeof etiology and availability 'of suitable drugs. A characteristic feapn-e in the development of thetherapy is the gradual c~e frqm the methods of manual rupture of the cysts, evaluated bY' ~'oberts (1955) and Dawson (1969), 'to the substitution; theral'Y, based on the prqduction or sexual hormones. ' Progesterone therapy' (Grandschamp 1960; Gamcik 1967; Gruss 1975)' was replaced by the use of hCG, frequently in combination with prog«sterone (Feustel 1974; Vrtel and KudIac 1976; Kudla/:: et al. 1977 and 1980). Recently, the therapy employs usually synthetichypothalamic gonadotropin-releasing hormones and prostaglandin analogues, also in combination (Kittock et al. 1973; Grunert and Ahlers 1974; Bentele and Humke 1979; Bostedt. et al. 1979; Bargai 1980; Stolla et al. 1980; Kudlac et a!. 1978, 1983, 1984). " , It ha1 been demonstrated that the syndrome of ovarian cysts in cattle is influenced by hereditary> factors (heritability hi = 0.30 to 0.35). Consequently, no bulls whose dams suffered repeatedly from cysts were approved for the use in artificial insemination. However, it can be, concluded lifter twenty years of using this rule ,that the occurrence of ovarian cysts under our conditions has. not decreased and remains at approximately the same level. Nevertheless, it is still Iiecessary to> include fertility in the selection criteria. High yielding dairy cows with very good fertility para-meters should be preferred (V (111991). , Many publications on the evaluation of the efficiency of the therapy by various drugs and by' -various therapeutic methods mentioned also the changes in genital organs and behaviour during • -the treatment. Recently, the study of the hormonal levels before, during and after the treatment was described by several authors (Bostedt et al. 1979; KudlBl! 1988; Grunert 1991). The _reliable information on the relationship between the syndrome of ovarian cysts and the blood -count before and after the treatment was, however, still missing. The economical aspects of the -treatment, concerning the optimum dosage and repeated administrations, are also very important. 'The present study deals with the above-mentioned two viewpoints of the problem. Materials and Methods The first part of the presented work deals with the effect of the therapeutical methods using 'synthetic GnRH, namely the locally produced commercial preparations DIRIGESTRAN inj. Spofa (control group), and SUPERGESTRAN inj. Spofa in the doses of 2 mI, and 4 mI im., crespectively, altogether in 244 cows. DIRIGESTRAN inj. Spofa contains synthetic hypothalamic hormone gonadorelin (LHRH, -4Ollgin 1 mI). We have demonstrated previously (Kudlal! etal. 1983; KudlBl! and Pichova 1990) -that after its administration a release of LH appears. A substantial increase of LH concentrations in blood can be detected by 30 minutes and lasts for 120 to 240 minutes. SUPERGESTRAN inj. Spofa contains 25 pg of GnRH analogue in 1 mI. Compared to the -previous preparation, the compound has a different structure of the synthetic decapeptide. Con• sequently, the biological effect was approximately two times stronger and was prolonged to the minimum of 240 minutes. Both preparations were administered im. to the cows of the Czech :Red and White breed and its crosses with Black and White and Holstein-Friesian breeds. The .oage of the cows was 3 to 7 years (first to fifth lactation). The cows had clinically detected syndrome -of ovarian cysts, found 60 to 90 days (exceptionally also by 120 days) after parturition. The cows were housed at the farms in the region of South Moravia, under comparable conditioqs of nutrition, housing, and husbandry. Their average yearly milk production was 4,000 to 5,000 kg. Altogeother 50 cows were treated with DIRIGESTRAN.inj. Spofa, and 194 cows with SUPERGESTRAN inj. Spofa (totally 244 animals). The therapeutical effect was evaluated after 10 to 14 days by :"8 complete gynaecological examination. The fertility of the treated cows was evalUated according -to the regular reproductive parameters (pregnancy rate after 1st insemination and all insemina-tions, insemination index, and the time period from the beginning of the treatment to the suc·cessful insemination). The results were evaluated statistically by PeaI:son's Xl test. The second part of the presented work consists of the study of the red and white blood cell Table 1 "The efficiency of the therapy of ovarian cyste In cows with hypothalamic GnRH (DIRIGESTRAN and SUPERGESTRAN Spofa)

The problem of ovarian cyst syndrome in cattle 'is being solved in veterinary medicine for~ decades.Therapy has been the main point for the research, with respect to the current knowledgeof etiology and availability 'of suitable drugs.A characteristic feapn-e in the development of thetherapy is the gradual c~e frqm the methods of manual rupture of the cysts, evaluated bY' ~'oberts (1955) and Dawson (1969), 'to the substitution; theral'Y, based on the prqduction or sexual hormones.' Progesterone therapy' (Grandschamp 1960;Gamcik 1967;Gruss 1975)' was replaced by the use of hCG, frequently in combination with prog«sterone (Feustel 1974;Vrtel and KudIac 1976;Kudla/:: et al. 1977 and1980).Recently, the therapy employs usually synthetichypothalamic gonadotropin-releasing hormones and prostaglandin analogues, also in combination (Kittock et al. 1973;Grunert and Ahlers 1974;Bentele and Humke 1979;Bostedt. et al. 1979;Bargai 1980;Stolla et al. 1980;Kudlac et a!. 1978Kudlac et a!. , 1983Kudlac et a!. , 1984))." , It ha1 been demonstrated that the syndrome of ovarian cysts in cattle is influenced by hereditary> factors (heritability hi = 0.30 to 0.35).Consequently, no bulls whose dams suffered repeatedly from cysts were approved for the use in artificial insemination.However, it can be, concluded lifter twenty years of using this rule ,that the occurrence of ovarian cysts under our conditions has.not decreased and remains at approximately the same level.Nevertheless, it is still Iiecessary to> include fertility in the selection criteria.High yielding dairy cows with very good fertility para-meters should be preferred (V (111991)., Many publications on the evaluation of the efficiency of the therapy by various drugs and by' -various therapeutic methods mentioned also the changes in genital organs and behaviour during • -the treatment.Recently, the study of the hormonal levels before, during and after the treatment was described by several authors (Bostedt et al. 1979;KudlBl! 1988;Grunert 1991).The _reliable information on the relationship between the syndrome of ovarian cysts and the blood -count before and after the treatment was, however, still missing.The economical aspects of the -treatment, concerning the optimum dosage and repeated administrations, are also very important.'The present study deals with the above-mentioned two viewpoints of the problem.

Materials and Methods
The first part of the presented work deals with the effect of the therapeutical methods using 'synthetic GnRH, namely the locally produced commercial preparations DIRIGESTRAN inj.Spofa (control group), and SUPERGESTRAN inj.Spofa in the doses of 2 mI, and 4 mI im., crespectively, altogether in 244 cows.
DIRIGESTRAN inj.Spofa contains synthetic hypothalamic hormone gonadorelin (LHRH, -4Ollgin 1 mI).We have demonstrated previously (Kudlal! etal. 1983;KudlBl! and Pichova 1990) -that after its administration a release of LH appears.A substantial increase of LH concentrations in blood can be detected by 30 minutes and lasts for 120 to 240 minutes.
SUPERGESTRAN inj.Spofa contains 25 pg of GnRH analogue in 1 mI.Compared to the -previous preparation, the compound has a different structure of the synthetic decapeptide.Con-• sequently, the biological effect was approximately two times stronger and was prolonged to the minimum of 240 minutes.Both preparations were administered im. to the cows of the Czech :Red and White breed and its crosses with Black and White and Holstein-Friesian breeds.The .oage of the cows was 3 to 7 years (first to fifth lactation).The cows had clinically detected syndrome -of ovarian cysts, found 60 to 90 days (exceptionally also by 120 days) after parturition.The cows were housed at the farms in the region of South Moravia, under comparable conditioqs of nutrition, housing, and husbandry.Their average yearly milk production was 4,000 to 5,000 kg.Altogeother 50 cows were treated with DIRIGESTRAN.inj.Spofa, and 194 cows with SUPERGESTRAN inj.Spofa (totally 244 animals).The therapeutical effect was evaluated after 10 to 14 days by :"8 complete gynaecological examination.The fertility of the treated cows was evalUated according -to the regular reproductive parameters (pregnancy rate after 1st insemination and all insemina--tions, insemination index, and the time period from the beginning of the treatment to the suc-•cessful insemination).The results were evaluated statistically by PeaI:son's Xl test.
The second part of the presented work consists of the study of the red and white blood cell  • count in the cows suffering from the syndrome of ovarian cysts and the changes of these parameters after the treatment.The study was conducted in the group of 40 cows out of the conditions as mentioned above.
The diagnostics was again based on the complete gynaecological examination.In selected cases additional laboratory examinations were performed: physical examination of cervical mucus, metabolic tests, and progesterone determination.
The study was conducted in the cows with syndrome of ovarian cysts occurring 60 to 90 days (exceptionally also by 120 days) after the parturition.
Blood samples from the selected cows were collected from vena jugularis.The hormonal treatment was scheduled subsequently.During the following days the cows were monitored for the occurrence of heat.Then 21 to 25 days after the treatment the cows were again examined gynaecologically, and blood samples were again collected .. Cows in heat were bred.The cows with negative palpatory finding in the ovaries, and pregnant after Ist or 2nd insemination, were considered as successfully treated.
Blood samples were collected from vena jugularis into two.test tubes with natrium citrate.Red blood cell and white blood cell counts were determined with the use of Btirker's chamber.
The white blood cells were differentiated in the panoptically stained smear.Haemoglobin was determined colorimetrically by cyanhaemoglobin method with the use of Spekol photocolorimeter (Germany).Hematocrit was evaluated after centrifugation (5 minutes at 15,250) in Wintrobe's capillaries.Sedimentation was examined in Westergreen's sedimentation instrument, adjustable to the angle of 60°.The other parameters of red blood cells were also calculated.Serum protein • fractions were determined by ele~ophoresis.The results were statistically evaluated by Student's t-test.

Results and Discussion
The results of the treatment of ovarian cysts with synthetic hypothalamic GnRH preparations DIRIGESTRAN inj.Spofa and SUPERGESTRAN inj.Spofa are s1::trmarized in Table 1.It can be concluded that the administration of DIRIGESTRAN inj.Spofa in the doses of 5 or 10 ml resulted in more or less the same effect as after the administration of 4 ml of SUPERGESTRAN inj.Spofa.The results of therapy with 2 ml of SUPERGESTRAN are worse in all evaluated parameters, and significantly worse in the percentage of successfully treated animals.
The number of animals, in which no ovarian cysts were found 10 to 15 days after the administration of 2 ml of SUPERGESTRAN, was lower by 23.9% in comparison to the group treated.with 5 and 10 ml of DIRIGESTRAN, and by 26.9% in comparison to the group treated with 4 ml of SUPERGESTRAN.

•
Pregnancy rates after the 1st insemination were lower in the group of animals treated with 2 ml of SUPERGESTRAN in comparison to the groups treated with DIRIGESTRAN (5 or 10 ml) or SUPERGESTRAN (4 ml).The difference was 8.62% and 6.07%, respectively.This result was not statistically significant, however, it should be considered that the number of animals was low.Similar results were obtained in the evaluation of pregnancy rates after all inseminations.Lower pregnancy rates were obtained in the group treated with 2 ml of SUPER-GESTRAN inj.Spofa but the differences were not statistically significant.
The time period from the first treatment until the insemination was practically the same in all groups, as well as the time period until the successful insemination.It can be concluded that the average time of 44.2 days required for getting the cow pregnant is economically feasible, and the described therapy schedule can be recommended.Stolla et al. (1980) studies also the therapeutical effect with the use of different preparations.The authors achieved 60% pregnancy rate after the treatment of • ovarian cysts with RECEPTAL Hoechst (dose of 10,ug GnRH), and 65% preg-nancy rate with the dose of 20 p,g.The differences were not statistically significant.• Our results correspond with the findings of Bargai (1982), who obtained the following results in the animals treated with oil, solution of LH and without the manual rupture of the cysts: 46.4 days from the treatment to the successful insemination, 65% of successfully treated animals, totally 47 % pregnant animals).In our previous works (Kudlac 1984) we obtained with LUTAL Hoechst 70 % success rate in the treatment of ovarian cysts and total pregnancy rate over 80% on average in 45 days after the treatment.The comparison demonstrates the similar results of therapy.Some differences in the results might originate from the different fertility of the experimental groups housed under various' conditions.
We attempted to determine the relation between the age of the cows at the treatment and the resulting effect.The average age of the cows before the treatment was 4.2 years.We have found that the successful treatment was achieved in the group of animals of the average age of 3.63 years.The age of the animals that were treated unsuccessfully averaged 5.50 years.The difference was statistically significant (P < 0.01). ,.
• Similar results were obtained in the evaluation of the group of cows according to the number of previous calvings.The overall average in the group was 2.076 previous calvings, with the average of 1.630 in successfully treated and 3.083 in unsuccessfully treated animals.The differences were significant between the whole group and the successfully treated group (P < 0.05) and highly significant between the successfully and unsuccessfully treated group (P < 0.01; Student's t-test).
Many authors studied the age and the number of previous calvings in the cows suffering from the syndrome of ovarian cysts, with regard to the relation to the gradually increasing milk production with the peak' between 4th and 7th year of age (2nd to 5th lactation).Kudlac et al. (1984) and Bargai (1982) mentioned that the syndrome of ovarian cysts occurred in 70% of the cases in the cows until 5 years of age.Bostedt et al. (1980) found that 33.6% of the cases of ovarian cysts occurred in cows at the age of 2 to 4 years.We have found no data ih the available sources on the evaluation of the relation of the age of the cows suffering from ovarian cysts and the therapeuti~l effect.It can be therefore concluded from' our results that the therapy with, GnRH has better prognosis in the younger animals with better adaptation possibilities that have to be always considered under the conditions of large-scale farms.
•   • Concerning red blood cell count there were no statistically significant differ-• ences (Tab.3a to 3c) among the groups of animals before the treatment, successfully and unsuccessfully treated.
Concerning white blood cell count there were no statistically significant differences between the numbers of white blood cells in the animals before the treatment and the groups of animals treated both successfully and unsuccessfully (Tab.4a).
The percentage of neutrophil granulocytes with segmented nucleus differed in the animals before the treatment and after the treatment, regardless to the result of the therapy.The difference between the animals before the treatment and the successfully treated animals was statistically significant (32.700 and 35.857;P < 0.05).The difference between the animals before the treatment and treated unsuccessfully (32.700 and 35.865) was not much distinct but not statistically significant, probably because of the small number in the group of unsuccessfully treated animals.
A similar pattern was found also in the case of neutrophil granulocytes with non-segmented nucleus.There was also an increase of the numbers of this type • of white blood cells after the treatment, regardless to its result.The statistical evaluation was similar as previously, i. e. significant difference between the groups before the treatment and successfully treated (4,106 and 5,112; P < 0.05) and a similar but not significant difference between the groups before the treatment and unsuccessfully treated (4,106 and 5,016).It can be assumed that the low number of unsuccessfully treated animals should be taken into account also in this case. .The relative content of lymphocytes (Tab.4b) changed markedly only in successfully treated cows (54,042 vs. 48,511).The decrease was statistically highly significant (p < 0.01).The difference between the groups of animals before the treatment and unsuccessfully treated (54,042 and 51,853) was statistically not significant.In the absolute numbers of lymphocytes the difference was 4,179 vs. 4,088 (before the treatment vs. unsuccessfully treated; the difference of more than 2% in the relative counts originated only from the calculation scheme).

Table 4a
Statistical sila.l.ftcance of the difrerea_ in white blood cell COUIlt in cows with ovarian cysts before and .fter the treatment the pregnancy.We mentioned in our previous works a possible connection • between the changes of white blood cell count and primary disorders of genital organs, and the changes in-hormone levels.In this work we attempted to study the relation between the syndrome of ovarian cysts and blood cell .counts.We recorded• the increase of the relative number of neutrophil granulocytes with segmented and non-segmented nucleus in treated animals.However, no clear relation to• the result of the treatment was established.Although we did not determine the levels of gonadal hormones in blood, it can be supposed that in successfully treated cows the influence of progesterone prevailed.It seems that the hormonal imbalance has no reflection in white blood cell count, namely in the relative proportion of separate types of leukocytes.The influence of other environmental factors, or stress situations at blo9d sampling, cannot be completely tuled out. .: A significant decrease of lymphocyte numbers occurred in treated cows, both in relative and absolute numbers.The difference is clear when the group of ammals before the treatment was compared also to the unsuccessfully treated cows.This finding corresponds to undoubtedly decreased level of oestrogens • in blood that appears after the ovarian cysts have disappeared.

Table 1 "The efficiency of the therapy of ovarian cyste In cows with hypothalamic GnRH (DIRIGESTRAN and SUPERGESTRAN Spofa) ,
I

Table 2
The efficiency of the therapy of ovarian cysts in cows with regard to age and number of previous calvings Table3a Statistical sipificance of the cWrer .... ces in red blood cell count in cows with ovarian, cysts before and after the treatm .... t ~ 2.730 75 "< !1,630 t,., ~.1.96019,• Table 3bStatistical significance of the cWrereDces in red blood cell count in cows with ovariaa cysts before and after the treatm .... t Table3c Statistical significance of the cWrer .... ces in red blood cell count in cows with ovarian cysts before and after • the treatm .... t " ..