ࡱ> |~{`bjbj.>\\\\\\\p4444tL5Tpm6(6666777JmLmLmLmLmLmLmnh qfLm]\&;77&;&;Lm\\66mccc&;\6\6Jmc&;Jmcc\\c66 k604W cvem0mcsrZcsrcsr\c7]8|c8d=9777LmLmc 777m&;&;&;&;ppp&+ ppp+ppp\\\\\\ Monitoring of early pregnancy and early embryonic mortality by ultrasound and determination of PAG and progesterone in cows N. Prvanovi1*, A. Tomaakovi1, J.F. Beckers2, J. Sulon2, T. Dobrani1, J. Grizelj1, P. Ko ila3, 1Clinic for obstetrics and reproduction, Faculty of veterinary medicine, University of Zagreb, Zagreb, Croatia 2Department for physiology of reproduction, Faculty of veterinary medicine, University in Liege, Liege, Belgium 3Veterinary station akovec, Croatia Abstract The aim of study was to investigate the role of pregnancy associated glycoproteins (PAG) in early pregnancy as well as possibility of using PAG and ultrasound as diagnostic tools in diagnosis of embrionic mortality. Our research included 74 simmental cows, 3-7 years old, calved every year. According to ultrasound findings (17., 24., 35. and 45. day after artificial insemination; AI) the cows were divided in 3 groups: pregnant cows (n=34), nonpregnant cows (n=18) and cows suffered embryonic mortality (n=21). Blood samples were collected every 72 hours between 12. and 45. day after AI and level of progesterone and pregnancy associated glycoproteins (PAG) were determined. Statistical analysis of variance for progesterone at day 12., 21. and 35. after AI shown significant difference between pregnant cows and both nonpregnant and embryonic mortality group (p>0,05). PAG variance analysis at day 24., 30. and 34. after AI shown high significant difference (p>0,01) between the nonpregnant and both embryonic mortality and pregnant group. On the other hand, variance analysis shown that middle values for PAG at days 40. and 45. after AI were highly significantly different (p>0,01) between pregnant and both nonpregnant and embryonic mortality group. The conclusion came out that it is impossible to determine embryonic mortality just on basis of progesterone profile, but it is easy to distinguish pregnant from nonpregnant cows, if cows are supposed to be pregnant more than 21 day. It is very easy and accurate to distinguish nonpregnant cows from cows that suffered early embryonic mortality. Furthermore, 98% cows in our research who experienced embryonic mortality, lost embryo 17-24 days after AI, visible in drastic decrease of PAG 7,5-9 days later. Using PAG for pregnancy diagnosis enables us to prove existence of alive vital embryo in uterus 24. days after conception. Key words: cow, early embrionic mortality, PAG, progesterone, ultrasound Introduction A variety of different methods have been, and still are used to detect the presence or absence of pregnancy in the cow. These range from the identification of substances that are present in body fluids using laboratory assays, and from different ultrasound modes, to simple clinical methods such as transrectal palpation; that is ubiquitously used method for the last 70 years, together with widespread management methods (failure to return to oestrus). In terms of efficient livestock production, it is the early identification of the nonpregnant cow that is important, since immidiate measures can be taken to attempt to induce pregnancy again as soon as possible. Realtime B-mode grey-scale ultrasound scanning is the method of choice for the early diagnosis of pregnancy in the cow. Using a 7.5 MHz transducer, Boyd et al. (1988) were able to confirm pregnancy as early as 9 days, whilst Pierson and Ginther (1984), using a 5 MHz transducer were able to do so at 12 and 14 days in heifers. However, experienced persons can accurately diagnose pregnancy, either at the time of, or before, the expected date of return to estrus in the nonpregnant cyclical animal. Accurate proof of existence alive and vital embryo, according to ultrasound findig could be made at day 24. when heart rate becomes visible (Goddard et al., 1994). There are also some laboratory methods widely used for early pregnancy diagnosis. The most common is determination of progesterone in blood and milk, 21 days after oestrus and determination of different pregnancy specific proteins, used in pregnancy diagnosis of ruminants till early ninetees (Zoli et al., 1992., Beckers et al. 1995., Szenci et al., 1996., Karen et al., 2002., Prvanovi et al., 2003). The main advantage of pregnancy specific proteins (PSP) is their ability to prove existence of placentation and presence of alive vital embryo, while progesterone only proves existence of corpus luteum. *dr. sc. Nikica Prvanovi, Clinic for obstetrics and reproduction, Heinzelova 55, 10 000 Zagreb, Croatia The most commonly used pregnancy specific protein for pregnancy diagnosis in cows is PAG (pregnancy associated glycoprotein).Pregnancy associated glycoprotein has been found in the serum of pregnant cattle and used as a pregnancy marker (Perenyi, 2002). As pregnancy failure occurs, PAG concentrations dropped and disappeared from maternal blood. Sequential assays of these proteins between day 24 and term are useful for studying the course of pregnancy, although it does not allow discrimination between early embryonic mortality and non-fertilization (Mialon et al., 1993). As PAG molecules are the products of the trophoblastic cells, it was suggested that their determination in maternal blood can be useful for prediction of fetal well-being and help to detect early placental abnormalities, embryonic mortality or abortion (Ectors et al., 1996.). Heifers expressing pregnancy failure between days 16 and 35 after AI have shown wide variation in pregnancy specific protein 60 (PSP60) concentrations at days 28 and 35. This phenomenon was explained by the variation in the time of embryo death (Mialon et al., 1993). This author reported some cases where at day 50 ultrasonic scanning and signs of oestrus indicated that the embryo was already dead, however till this day the PSP60 concentrations were increasing. Decreasing PSP60 concentrations were observed in some animals before day 50, were according to ultrasonic examination, the conceptus was still alive. Pregnancy specific protein B ( BPSPB) test in conjunction with milk progesterone test was used to determine the time of the embryonic death (Humblot et al., 1988). In most of the early failures an elevation in the PSPB concentrations was not detectable, or only after day 24. No coefficient of correlation between PSPB and progesterone concentration was significant at any days of gestation studied. Significant difference was found in the rate of mean PSPB levels between pregnant cows and cows showing late embryonic mortality between days 24 and 30-35 after AI. In their study it was not possible to predict the occurrence of embryonic mortality. After invitro production of embryos or cloning, the PAG follow up in plasma samples collected weekly was able to monitor embryonic or fetal mortality (Ectors et al., 1996, 1997). In that studies it was concluded that significantly increased PSP60 levels could indicate abnormal placental development. In veterinary practice, PAG and PSPB RIAs in biological fluids are helpful to confirm clinical diagnoses established by ultrasonography (Szenci et al., 1998.) Cows presenting an initial positive and then negative ultrasonographic diagnosis expressed decreasing PAG concentrations (Szenci et al., 1998) The high progesterone and PAG concentrations confirmed the presence of a live conceptus ansd a functional corpus luteum at the time of the first ultrasonographic examination. After the time of embryonic mortality occurred, the PAG levels had already decreased, while progesterone concentrations have shown that the corpus luteum was maintained in three of four cases. Following embryonic mortality using ultrasonography, PAG and PSPB concentrations decreased steadily, in one animal these protein concentrations did not reach the threshold level (Szenci et al., 1998). Mentioned authors were able to diagnose late embryonic and fetal mortality cases retrospectively in seven cows using PSPB RIA test and in four cows using PAG RIA test (Szenci et al., 2000). Because of the large individual variations in PAG concentrations in maternal blood, only the marked decrease or disappearance in serum/plasma concentrations of these proteins can be a no controversial, predictive sign of embryonic or fetal death. Material and methods: Our research included 74 simmental cows, 3-7 years old, which were regularly used in reproduction and calved every year. According to ultrasound findings (17., 24., 35. and 45. day after artificial insemination; AI) the cows were divided in 3 groups: pregnant cows (n=34), nonpregnant cows (n=18) and cows suffered embryonic mortality (n=21). Cows which were suspect to be pregnant by all four examinations were divided into pregnant group. Cows who were considered to be pregnant according to first or first two ultrasound findings and nonpregnant according to the last or last two findings were divided into embryonic mortality group. Cows considered to be nonpregnant by all four ultrasound findings were divided into nonpregnant group. Blood samples were collected every 72 hours between 12. and 45. day after AI and level of progesterone and pregnancy associated glycoproteins (PAG) were determined. We used only sampligs which could be diagnostically valuable. For progesterone at days 14., 21. and 35. after AI and for PAG at days 24., 30., 34., 40. and 45. after AI. The PAG measurements were performed according to the method of Zoli et al. (1992) with some modifications. Briefly, the previously weighed standards and the plasma samples (0,1 ml) were diluted in 0,2 ml of Tris buffer at pH 7.5 {0,025 M Tris, 0,01 M MgCl2 0,01% (wt/vol) sodium-azide, 0,05% (wt/vol) bovine serum albumin (BSA). The standard curve ranged from 0,2 to 25 ng/ml and in order to minimize non-specific interference 0,1 ml virgin heifer plasma was added to each tube of the standard curve. After the addition of appropriate dilutions of antisera the plasma samples and the standards were incubated overnight at room temperature. Antisera titres were determined to obtain a tracer binding-ratio in the zero standards of approximately 20-30%. The following day, tracer (0,1 ml or 25 000 cpm) was added to all the tubes and the tubes were incubated for 4 h at room temperature. The total assay volume was 0,5 ml. The separation of the free and bound fractions was carried out by centrifugation ( 20 min at 1500xg, 10C) after the addition of 1 ml second antibody polyethylene glycol (PEG) solution {0,17% (v/v) normal rabbit serum, 0,83% (v/v) sheep anti-rabbit IgG, 0,3% (wt/vol) BSA, 4% (wt/vol) polyethylene glycol 6000 in Tris buffer}. After the tubes had been incubated for 1 h with the second antibody PEG solution, 2 ml of Tris buffer was added and the tubes were centrifuged (20 min at 1500xg). The supernatant was aspirated and 4 ml of Tris buffer was added to each tube. The tubes were centrifuged (20 min at 1500xg) and the supernatant aspirated. The 125I in the pellet was quantified using a gamma counter (LKB Wallac 1261 Multigamma counter,LKB, Turku, Finland) with a countig efficeiency of 75%. Level of progesterone in sera samples was determined using standard RIA method with conjugated steroids (Coat-A-Count TKPG, Diagnostic product Corporation) without preincubation. The 125I in the pellet was quantified using a gamma counter (LKB Wallac 1261 Multigamma counter,LKB, Turku, Finland) with a countig efficeiency of 75%. All samples in both methods (PAG and progesterone) were analysed in duplicate with middle value as result and all suspect results were repeated. We used program Statistica and ANOVA to perform variance analysis and linear correlations. Results: Abbrevitaions used in results: PAG1- sampling at day 24.after AI PAG2- sampling at day 30. after AI PAG3- sampling at day 34. after AI PAG4- sampling at day 40. after AI PAG5- sampling at day 45. after AI PROG1- sampling at day 14. after AI PROG2- sampling at day 21. after AI PROG3-sampling at day 35. after AI Table 1: Descriptive statistics for group of pregnant cows Valid NMeanMinimumMaximumVarianceStd.Dev.Standard ErrorPAG1320,8946560,31,7840,0819590,2862850,050609PAG2321,4536880,7022,4360,1814490,4259680,075301PAG3322,1036881,3273,2340,3527490,5939270,104992PAG4322,7009691,565,2230,6061270,7785410,137628PAG5323,5200631,7675,8830,9073810,9525660,168391PROG1326,3796252,7619,6714,0182872,0045660,354361PROG2327,5871563,17812,3645,467642,3382990,413357PROG3328,8694064,60517,44510,415093,2272420,570501Table 2 Descriptive statistics for group of embryonic mortality cows Valid NMeanMinimumMaximumVarianceStd.Dev.Std.ErrorPAG1211,1155240,5741,8720,1009830,3177780,069345PAG2210,9050950,32,4360,269020,5186720,113183PAG3210,57714302,8650,8576240,926080,202087PAG4210,4198103,6391,1581631,076180,234842PAG5210,40503,9241,3724161,1715020,255643PROG1214,8764762,3648,563,5331351,8796630,410176PROG2211,009810,1651,8370,1789440,4230180,09231PROG3214,5923332,0169,3553,4457941,8562850,405075 Table 3 Descriptive statistics for group of nonpregnant cows Valid NMeanMinimumMaximumVarianceStd.Dev.Std.ErrorPAG1180,01116700,1240,0011210,0334840,007892PAG2180,01988900,3580,007120,0843810,019889PAG3180,00622200,1120,0006970,0263990,006222PAG418000000PAG518000000PROG1183,7614440,7925,851,4461961,2025790,283451PROG2181,0192220,1114,1930,9691160,9844370,232034PROG3214,1080480,2657,7942,1745921,474650,321795 Figure 1: PAG level in blood of pregnant, nonpregnant and embryonic mortality group in ng/ml (Values with different superscripts within the same columns differ significantly (P<0.05; ANOVA)  EMBED MSGraph.Chart.8 \s  Figure 2: Progesterone level in blood of pregnant, nonpregnant and embryonic mortality group in ng/ml (Values with different superscripts within the same columns differ significantly (P<0.05; ANOVA)  EMBED MSGraph.Chart.8 \s  Variance analysis has shown that there is significant difference (P<0,05) for middle values of PAG1, PAG2 and PAG3 between all three groups. Variance analysis for PAG4 and PAG5 has shown that there is high significant difference (P<0,01) between pregnant and both nonpregnant and embrionic mortality group. Variance analysis for PROG2 has shown significant difference (P<0,05) between pregnant cows and both nonpregnant and embrionic mortality group. Variance analysis for PROG 1 and PROG 3 didn't show any significant difference between the groups. Ananlysis of linear correlations between the group didn't show any significant linear correlation between the parameters or groups. Discussion and conclusions Statistical analysis of variance for progesterone at day 12., 21. and 35. after AI shown significant difference between pregnant cows and both nonpregnant and embryonic mortality group (p>0,05). PAG variance analysis at day 24., 30. and 34. after AI shown high significant difference (p>0,01) between the nonpregnant and both embryonic mortality and pregnant group. On the other hand, variance analysis shown that middle values for PAG at days 40. and 45. after AI were highly significant different (p>0,01) between pregnant and both nonpregnant and embryonic mortality group. The <DXZ   4 6 : R T h j n ~ ʼʼʤʼʼʏpppdphh9CJaJmHsHhh9hh9CJaJmHsHhh9hh9CJH*aJmHsHhh9H*mHsHhh9hnk_5mHsHhh9h5mHsHhh9h ~-5mHsHhh9h 5H*mHsHhh9h(5mHsHh(mHsHh(hn5mHsHhRY5mHsHh(h(5mHsH# ~ RFxzPRf#%'C+ $`a$gd ~-$a$gd&z d`gd6E $dha$gdh9$a$gd qH$a$gd(   5 6 U ` m r s w | <>?LMɺغغثغ؜؍غ؍غ؜~~~~hh9hhCJOJQJaJhh9hgCJOJQJaJhh9h(CJOJQJaJhh9h9MCJOJQJaJhh9h6ECJOJQJaJhh9h qHCJOJQJaJhh9hCJOJQJaJh qHh(5mHsHh qHh qH5mHsH/;<Y`pqwĵĦⵗyj_TEhh9h'&CJOJQJaJhh9h'&CJaJhh9h`w~CJaJhh9hCJOJQJaJhh9h"cCJOJQJaJhh9h`w~CJOJQJaJhh9hhCJOJQJaJhh9hgCJOJQJaJhh9h6ECJOJQJaJhh9h qHCJOJQJaJhh9hRYCJOJQJaJhh9hT/CJOJQJaJhh9hybCJOJQJaJ*`insuv+0OP%&pqQRĵӌӌ}}}}}}}}qhCJOJQJaJhh9hCJOJQJaJhh9h9MCJOJQJaJhh9h{CJaJhh9hCJOJQJaJhh9h"cCJOJQJaJhh9hgCJOJQJaJhh9hRYCJOJQJaJhh9hCJOJQJaJhh9h{CJOJQJaJ,R[]EFIJżxgVgEg hfAh|CJ^JaJmH sH  hfAhc;lCJ^JaJmH sH  hfAhvACJ^JaJmH sH hfAhdCJaJmHsHhfAhCJaJmHsHhfAht5CJaJmHsHhfAh/ZCJaJmHsHh ~-mHsHhRY5mHsHh/Z5mHsHh ~-5mHsHhfAhfACJaJmHsHhfAhfA5CJaJmHsHhfA5mHsHDPvx|"$(PR&XQ C!D!N!!޼zk\M>/>hfAhnk_CJaJmH sH hfAhH iCJaJmH sH hfAh7"UCJaJmH sH hfAh9kCJaJmH sH hfAho}CJaJmH sH hfAh8YCJaJmH sH hCJaJmH sH hfACJaJmH sH hfAhfACJaJmH sH hfACJaJmH sH  hfAhdCJ^JaJmH sH  hfAhH iCJ^JaJmH sH  hfAh8YCJ^JaJmH sH  hfAhs?CJ^JaJmH sH ! "e#f#### $%%&&&''(B+C+:,O,P,..//*0ĵĵĵwhwYJhfAhTCJaJmH sH hfAh]CJaJmH sH hfAhBrCJaJmH sH hfAh|iPCJaJmH sH hfAh7"U5CJaJmH sH hfAh|iP5CJaJmH sH hfAh%)CJaJmH sH hfAh_CJaJmH sH hfAhnk_CJaJmH sH hfAhlCJaJmH sH hfAhH iCJaJmH sH hfAh7"UCJaJmH sH C+:,P,w9x9999 :.:Q:u::::::::; ;; $IfgdPbe$a$gd4S$L^L`a$gd $ ^ a$gd$a$gd$a$gde $a$gd&z*0I00`11111p3 44 6u6667779Ĵĥ▇xiYiJ:JhfAhe CJH*aJmH sH hfAhe CJaJmH sH hfAhXCJH*aJmH sH hfAhXCJaJmH sH hfAhH/CJaJmH sH hfAh!GvCJaJmH sH hfAhvCJaJmH sH hfAhBrCJaJmH sH hfAhZBCJH*aJmH sH hfAhZBCJaJmH sH hfAh]CJaJmH sH hfAhTCJaJmH sH hfAhtPCJaJmH sH 9v9w9x999999999 ::-:2:P:V:t:z::::::::::ⷧzj[KhfAhPbe5CJaJmH sH hfAhPbeCJaJmH sH hhPbe5CJaJmH sH hfAh4SCJaJmH sH hfAh CJaJmH sH hhCJaJmH sH hh5CJaJmH sH hCJaJmH sH hfAh|iP5CJaJmH sH hfAhXCJaJmH sH hfAhe CJaJmH sH hfAhf8CJaJmH sH :::;;;C;L;q;r;z;;;;;;;;;;<<$<-<T<U<^<g<<<<<<<<<== =I=J=K=====׹|n|nhh 5CJ^JaJhh CJ^JaJ hh CJ^JaJmH sH hh(5CJaJmH sH hh CJaJmH sH hh 5CJaJmH sH hhPbe5CJ^JaJhhPbeCJaJhhPbeCJ^JaJ hhPbeCJ^JaJmH sH +;;";+;4;:;;;@;C;L;P;V;_;h;q;r;w;z;;;;;;;;;FfFf $$Ifa$gdPbeFfS $IfgdPbe;;;;;;;;;;;;;< <<<<!<$<-<3<9<B<K<T<U<FfFfFfW $$Ifa$gdPbeU<[<^<g<m<s<|<<<<<<<<<<<<<<<<<<<<=FfFf[ $$Ifa$gdPbe==I=J=K=S=X=`=h=q=z== $IfgdgV$a$gd&zgde Ff ==kd !$$Iflִ, g0!'''S';'v'S'S'S0!6    44 la]========= $$Ifa$gdgV $IfgdgV==kd"$$Iflִ, g0!'''S';'v'S'S'S0!6    44 la]========= $$Ifa$gdgV $IfgdgV==kd#$$Iflִ, g0!'''S';'v'S'S'S0!6    44 la]===>> >>>'> $$Ifa$gdgV $IfgdgV==>0>8>c>i>>>>>??8?:?(>kd#$$Iflִ, g0!'''S';'v'S'S'S0!6    44 la](>->0>8>:>@>I>Q>Z> $$Ifa$gdgV $IfgdgVZ>[>kd$$$Iflִ, g0!'''S';'v'S'S'S0!6    44 la][>`>c>i>k>q>z>>> $$Ifa$gdgV $IfgdgV>>kd%$$Iflִ, g0!'''S';'v'S'S'S0!6    44 la]>>>>>>>>> $$Ifa$gdgV $IfgdgV>>kd&$$Iflִ, g0!'''S';'v'S'S'S0!6    44 la]>>>>>>>>> $$Ifa$gdgV $IfgdgV>>kd'$$Iflִ, g0!'''S';'v'S'S'S0!6    44 la]>?????%?.?7? $$Ifa$gdgV $IfgdgV7?8?kd($$Iflִ, g0!'''S';'v'S'S'S0!6    44 la]8?9?:?;?!'''S':'v'S'S'S0!6    44 la]????????? $$Ifa$gdgV $IfgdgV??kd*$$IflSִ, u>!'''S':'v'S'S'S0!6    44 la]?????@ @@@ $$Ifa$gdgV $IfgdgV@@kd+$$IflSִ, u>!'''S':'v'S'S'S0!6    44 la]@$@'@0@2@8@A@J@S@ $$Ifa$gdgV $IfgdgVS@T@kd,$$IflSִ, u>!'''S':'v'S'S'S0!6    44 la]T@Y@\@^@`@b@d@f@h@ $$Ifa$gdgV $IfgdgVh@i@kd-$$IflSִ, u>!'''S':'v'S'S'S0!6    44 la]i@n@q@s@u@w@y@{@}@ $$Ifa$gdgV $IfgdgV}@~@kd.$$IflSִ, u>!'''S':'v'S'S'S0!6    44 la]~@@@@@@@@@ $$Ifa$gdgV $IfgdgV@@kd/$$IflSִ, u>!'''S':'v'S'S'S0!6    44 la]@@@@@@@@@ $$Ifa$gdgV $IfgdgV@@kd0$$IflSִ, u>!'''S':'v'S'S'S0!6    44 la]@@@A AAA A)A $$Ifa$gdgV $IfgdgV)A*Akd1$$IflSִ, u>!'''S':'v'S'S'S0!6    44 la]*A+AAA BBBBB%DEEEErst d`gdR$a$gdf8 $7$8$H$a$gdr$a$gd&zKAAAAAABBBB B BoBqBBBBp`pP;(jhfAh7+5CJUaJmHsHhfAhf85CJaJmH sH hfAhX5CJaJmH sH hfAhf85CJaJmH sH (j2hfAhr5CJUaJmH sH +jqH hfAhrCJUVaJmHsH(jhfAhf#5CJUaJmH sH hfAhf#5CJaJmH sH hfAhrCJaJmH sH hfAhr5CJaJmH sH hfAhf#5CJaJmH sH BBBBBBBCC:CC$D%DADEDbDkDDEEEEįqbqbbSbSbSbSDhfAhRCJaJmHsHhfAh8.CJaJmHsHhfAhaCJaJmHsHhfAh"?CJaJmHsHhfAh2CJaJmHsHhfAh25CJaJmHsHhfAhf#5CJaJmHsH(jhfAh7+5CJUaJmHsH(j@<hfAhM5CJUaJmHsH+jqH hfAhMCJUVaJmHsHhfAh7+5CJaJmHsHEEFFGGGrsƹhfAhB5CJaJmHsHhB5CJaJmHsHhR5CJaJmHsHUhfAhRCJaJhfAhRCJOJQJaJhfAhR5CJaJmHsH conclusion came out that it is impossible to determine embryonic mortality just on basis of progesterone profile, but it is easy to distinguish pregnant from nonpregnant cows, if cows are supposed to be pregnant more than 21 day. It is very easy and accurate to distinguish nonpregnant cows from cows that suffered early embryonic mortality. Furthermore, 98% cows in our research who experienced embryonic mortality, lost embryo 17-24 days after AI, visible in drastic decrease of PAG 7,5-9 days later. Using PAG for pregnancy diagnosis enables us to prove existence of alive vital embryo in uterus 24. days after conception. References: ,1h. A!"#$% Q$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,5Q92 22 22 22 22 22 22 22 24 Wkd$$If#ִB 5(y"'Q'Q'Q'Q'Q'Q'Q'Q0"6    2 22 22 22 22 22 22 22 24aQ$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,592 22 22 22 22 22 22 22 24 Wkd$$If#ִB 5(y"########0"6    2 22 22 22 22 22 22 22 24aQ$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,592 22 22 22 22 22 22 22 24 WkdX$$If#ִB 5(y"########0"6    2 22 22 22 22 22 22 22 24aQ$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,592 22 22 22 22 22 22 22 24 Wkd $$If#ִB 5(y"########0"6    2 22 22 22 22 22 22 22 24aQ$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,592 22 22 22 22 22 22 22 24 Wkd$$If#ִB 5(y"########0"6    2 22 22 22 22 22 22 22 24aQ$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,592 22 22 22 22 22 22 22 24 Wkd\$$If#ִB 5(y"########0"6    2 22 22 22 22 22 22 22 24aQ$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,592 22 22 22 22 22 22 22 24 Wkd$$If#ִB 5(y"########0"6    2 22 22 22 22 22 22 22 24aQ$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,592 22 22 22 22 22 22 22 24 Wkd$$If#ִB 5(y"########0"6    2 22 22 22 22 22 22 22 24aQ$$If!vh5Q5Q5Q5Q5Q5Q5Q5Q#vQ:V #0"6,592 22 22 22 22 22 22 22 24 Wkd`$$If#ִB 5(y"########0"6    2 22 22 22 22 22 22 22 24a$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5;5v5S5S5S#v#vS#v;#vv#vS:V l0!6,55S5;5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a]$$If]!vh555S5I5v5S5S5S#v#vS#vI#vv#vS:V lS0!6,55S5:5v5S9a] Dd n b  c $A? ?3"`?"\MEDg 2@=\MEDg $6`ErzxmhUyG#k̦mVu7Mbl37_tucG`  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`bcdefghijklmnopqrstuvwxyz}Root Entry F@60Data aDWordDocument.ObjectPooli60@60_1215419139 Fi60k60Ole CompObjjObjInfo  !"#$%&'()*+,-./0123456789:;<=>?ABCDEFGHJKLMNOQ FMicrosoft Graphov grafikonGBiff5MSGraph.Chart.89q  FMicrosoft Graphov grafikonGBiff5MSGraph.Chart.89q Workbook_1215414003 Fk60k60Ole EPRINT . B"#,##0\ "kn";\-#,##0\ "kn"##,##0\ "kn";[Red]\-#,##0\ "kn"$#,##0.00\ "kn";\-#,##0.00\ "kn")$#,##0.00\ "kn";[Red]\-#,##0.00\ "kn">*9_-* #,##0\ "kn"_-;\-* #,##0\ "kn"_-;_-* "-"\ "kn"_-;_-@_->)9_-* #,##0\ _k_n_-;\-* #,##0\ _k_n_-;_-* "-"\ _k_n_-;_-@_-F,A_-* #,##0.00\ "kn"_-;\-* #,##0.00\ "kn"_-;_-* "-"??\ "kn"_-;_-@_-F+A_-* #,##0.00\ _k_n_-;\-* #,##0.00\ _k_n_-;_-* "-"??\ _k_n_-;_-@_-1Arial1Arial1Arial1Arial1Arial1Arial1Arial=  ,##0.00\,ZR3 A@@   ` J ` J 883ffff̙̙3f3fff3f3f33333f33333\R3&STUPAG 1PAG 2PAG 3PAG 4PAG 5 pregnant cowsr?g)YNB?_Z@@F) @embrionic mortalitys/?fʉ?İØw? )"*?Q?nonpregnant cows*Z^ކ?X32]?WY(-= *>X4`H(  ~  <  @AA?! 2 ]  O $~~  <  @AA?  ]  O $<a< ~~  <d  @AA?  ]d  O $<b<~~  <  @AA? p ] 0 O $<b< h3dh 3Q  pregnant cowsQQQ3_4E4 3Q *embrionic mortalityQQQ3_4E4 3Q $nonpregnant cowsQQQ3_4E4 3QQQQ3_4E4 3QQQQ3_4E4 3QQQQ3_4E4 3QQQQ3_4E4D$% M3O&Q4$% M3O&Q4FAO  3OU  3 b#M43*#M! M4523  O43"  M3O t;% M3O&Q423 M NM44444 lmu>  4T EMF .*xQF, EMF+@XXF\PEMF+"@ @ $@ 0@?!@ @     !" !" !  Rp"Arial XX  /E0Ddv p>>\0gp ?0p =0pg`0 g0dv% % % % " !%   N % " !%   N '% %    V0ooo&% % % % " !% % %   ~p69 z   6o6o6o6o6o% % % " !% % %   N 9 z&% (   6o6o66% % % " !% % %   N &% (   66666666o*6*6*6*6o*6o% % % " !% % %    . % % % " !% % %   /% % % " !% % %   ,% ( &% (   676&% ( 66&% ( (66'% &% (   V0inn% % % " !% % %   ,7  V0Q"7L7"% % % " !% % %   ,  V0% % % " !% % %   ,'% ( &% (    +% % % " !% % %   ,  +% % % " !% % %   ,  +% % % " !% % %   ,('% ( &% (    V,B==% % % " !% % %   ,  V,% % % " !% % %   ,  V,,''% % % " !% % %   ,% % % " !% % %   /% % % " !% % %   N % % % " !% % %   N % % % " !% % %   O    TT8\@]4@LP0%TTm\@]4@mLP2%TT<\@]4@LP4%TTq\@]4@qLP6%TT@\@]4@LP8%TXmu\@]4@muLP10%%% % % " !% % %   N % % % " !% % %   O   TlgQP\@]4@gQLXPROG1-044%TlHQ1\@]4@HQLXPROG2-044%Tl)Q\@]4@)QLXPROG3-044%% % % " !% % %   N &% ( % (   +9 z% % % " !% % %   = v% % % " !% % %   = v&% ( o  6r o o'%   V0 U8  Z3 o  o Z   T Im \@]4@ Lhpregnant cows )%))%)%)5% % % % " !% % %   = v% % % " !% % %   = v&% (   6r  '% (   + 2    T  \@]4@ Ltembrionic mortality%<)))%<)%" % % % " !% % %   = v% % % " !% % %   = v&% ( E  6r E E'% (   V, +8 _ 03 Z Z 0   T  l\@]4@ ]Llnonpregnant cows))))%))%)%)5% % % % " !% % %   = v% % % " !% % %   N % ( % ( %  0  F(GDICFGDIC% %   % %   % % % % %   TT\@]4@LPa%% " !  N % % F(GDICFGDIC% %   % %   % % % % %   TT\@]4@LPb)% " !  N % % F(GDIC`FGDIC% %   % %   % % W% % %   TTF\@]4@LPb)% " !  N % % K@0 N N   % % " N !  " !  ( " F4(EMF+*@$??FEMF+@ CompObjjObjInfo Workbook 1Tablesr B"#,##0\ "kn";\-#,##0\ "kn"##,##0\ "kn";[Red]\-#,##0\ "kn"$#,##0.00\ "kn";\-#,##0.00\ "kn")$#,##0.00\ "kn";[Red]\-#,##0.00\ "kn">*9_-* #,##0\ "kn"_-;\-* #,##0\ "kn"_-;_-* "-"\ "kn"_-;_-@_->)9_-* #,##0\ _k_n_-;\-* #,##0\ _k_n_-;_-* "-"\ _k_n_-;_-@_-F,A_-* #,##0.00\ "kn"_-;\-* #,##0.00\ "kn"_-;_-* "-"??\ "kn"_-;_-@_-F+A_-* #,##0.00\ _k_n_-;\-* #,##0.00\ _k_n_-;_-* "-"??\ _k_n_-;_-@_-1Arial1Arial1Arial1Arial1Arial1Arial= < ,##0.00\ZR3 A@@   ` J ` J 883ffff̙̙3f3fff3f3f33333f33333\R3&STUPROG1PROG2PROG3pregnant cows ~j@ș&l?Y@q"!@embrionic mortalityd삁@҇.(?]^@nonpregnant cows&o@UN?ė"n@WYc3P= *>X4(  x  6 @AA?] V $<a<xx  6  @AA?] <V $<b<xx  64  @AA?8 ]4 V $<b<z~3d 3Q  pregnant cows QQQ3_4E4 3Q *embrionic mortalityQQQ3_4E4 3Q $nonpregnant cowsQQQ3_4E4D $% M3O&Q4$% M3O&Q4FA 3OQ7 3 b#M43*#M! M4523  O43"  '3O '% M3OQ423 M NM44444 H? FA$4Od%)?,!L<}}xysssk@ L 30W 9kk [x'5G~@!x|?cW@m"㶹xeUNMȇ+}U2+W%pJH3`4?X fq~B>SrGtuX62zY!J~'U{1gk ;Zj''5Xigg2¤SޑH  SMhL;@6׷C`ѸwȂX_sD˥Ngolq.Bl{C=}}I'W_^۾fΎmFڞh}/1j;Boaōl1Rc24N Sˑ=f7Iy S7vs'xL;\?00n~)LAŖ:[ 8!()3ZEX?/"; qΛ Gu3SNd7Kg\rT_՘^ L39^.b1麌 V*RsO0M3poUo7VDG-O`?s@⑪Fd|oV V*RL1c,L"ET'Ax"_q$%G?!Yzk@)9kᭋm.lb<\*gc$$?]<+5)2*2pXpЎ֍} QE"CT!wbkfTl7'GvUj?(U֔[ ͚D2L:gk:wk:t'uI^_kgc>ҵQ 'y ~ya0šA+"98bmbbYe/(G]}+5k˜},؟Av!Tq/k8ReIC::Q]: ŬvOX/"NPl:BGu'](. "a"=(8ot_<~/Xcz+f9*D3V5g:XNItb!Q\KaM?z<ߐKYaI! GMNQ2y$6L,w#{I?rbw'%/HSf^輕X"8[ܻ dBikh6Yin\ff Ӭ,qAs\\4*1>\w2n=q>8AuR+!é y"jdz4SU4Jt8"qzKy [^\ݙL?Mo^͉C JSw^%3t6nd'鼾ֈ zX単#~U"8ޥxC9[Ig8vaCX>#Vޭߓ~k?=͋}G4%n{%dxQ`TPה};G~]u *ͥ+V@_0cnJ3}7>׍;N<|# سF,_霧 y>wr8sN?g ƐbRg^Cg9N}njư0mBpw'h /^yx\CҚXkGg$ST!h96 }k'teL,`]PESMMcYO1y,U% dX$VefN')@p#E0xbGe̿{RAη\ .UHu#=c}x  i""|i8U[o{!:=X-b#H}g5#Snm{RǿuoyUfo]/l|-ϴ+M CCC`27QLq?j߈zُbMEЋ^U^sOO{0~&W妷 w79@1=i)21OИVqS$MW ||3ҳ_ҳ[8)~2xKC(YΧ?UeVOZpדo&O|BZK\VaWmwqK~P֓(z|6#$ruB^Bxh,g:UMy|a8@Xfo5G$6GGѐF>ڽ9.{K9y#m!{IΎgivQcZ単&Qsau/Kd:%,\Xӯҥ񶱟K'-q})rP ʨR)%OI,AY͐:b8EEϿd6(^*=^C@č53'OkvaisO[k.\˧?5ԏ@ ʧ>V}܌f|AK$>$F"}Aa1N\O@㨮Nk w*4ۙf v4Ьg-.4}\ O\4\*qg E>h6h6\i|K=D~~zƒy_jfe?F޳t=K k0]5lls\^F8m`I%6`if<pA'"t pUsQgԀN3}<Ӱ1VȱOX<ϠƽJ; sO\v^w@sv~K"`z;sIɋi|Ϋ}hSoA.ǚcXӭ^;CFXml>lٮ]0 #wwB!;ˤx mܧjrTޯ~O_-ߧ{ڗu*bv.ϗg,>>F8nQowLߎqy!ղs훉/*ˆq?^26|sh0Ƴņ}ذɴN2 ;~ eQ6k5H61xZBGտ)Tiem"o;M@]@ZB2=dS_2ASummaryInformation(@<DocumentSummaryInformation8ICompObjPsOh+'0 4@T`l|     Monitoring of early pregnancy and diagnostics of early embrionic mortality by ultrasound and determination of PAG and progesterone in cows nprvanovicNormal nprvanovic4Microsoft Office Word@6F@0xW0@H10@Љ60T ,5՜.+,0x hp  WRH-TDUqa> Monitoring of early pregnancy and diagnostics of early embrionic mortality by ultrasound and determination of PAG and progesterone in cows Naslov@@@ NormalCJ_HaJmH sH tHP@P &zNaslov 3$dh@&5OJQJ^JmHsH>A@> Zadani font odlomkaVi@V Obi na tablica4 l4a .k@. Bez popisabB@b  Tijelo teksta$7dh`7a$OJQJ\aJ(mHsH>|}OR F <=fC: P w-x---- ...Q.u......../ ///"/+/4/:/;/@/C/L/P/V/_/h/q/r/w/z/////////////////////0 0000!0$0-03090B0K0T0U0[0^0g0m0s0|0000000000000000000011I1J1K1S1X1`1h1q1z1111111111111111111111122 222'2(2-20282:2@2I2Q2Z2[2`2c2i2k2q2z222222222222222222222233333%3.3738393:3;3<3z3{3|33333333333333333333334 4444$4'4042484A4J4S4T4Y4\4^4`4b4d4f4h4i4n4q4s4u4w4y4{4}4~4444444444444444444445 555 5)5*5+555 66666%89999q>r>s>>>0000000000000000000000000000@0@0@0@0@0@0000000 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0000000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 000000000000000000|}Q.u..:/;/q/r/////00T0U0000000011111111'2(2Z2[22222223%3.3738393:3z3333344S4T4h4i4}4~4444455 5)5*5+556q>>{00!c{00{00X3{00t{00{00{00{00{0 0 {00{0 0 {00{00{00{00{00{00{00{00{00{00{00{00{00{00{00{00{00u@0 {0 0 u@0{0 0 u@0{00u@0{{00u@0{{00u{00{00u@0{00u@0{0203( t{020 {020 {00 @0{00{00{00{00@ɛ @ {0 0 @0'0:{0 0 @0{00@10{{00@ @0{{00{00L,{00@0{00@0{0K0LԷt{0K0 {0K0{00{Az10H0H@0H{000H R!*09:=KABE$'()*+,./0;Z[\C+;;U<======='>(>Z>[>>>>>>>7?8?????@@S@T@h@i@}@~@@@@@)A*A%-123456789:<=>?@ABCDEFGHIJKLMNOPQRSTUVWXY&566666>::8@0(  B S  ? V  W VXYZ[ĥ\] ^<_F` KaTKz%(S) + ++X,X,_,>   %(Z)+++],f,f,>  C *urn:schemas-microsoft-com:office:smarttagsmetricconverterB *urn:schemas-microsoft-com:office:smarttagscountry-regionh *urn:schemas-microsoft-com:office:smarttagsCity0http://www.5iamas-microsoft-com:office:smarttagsV *urn:schemas-microsoft-com:office:smarttagsplacehttp://www.5iantlavalamp.com/ Y~0,01 M10C6000 in ProductID    "'(12;<>?IN[gsw{ )3[^_imy{()34AKPQZ^bfquz !+,4LOPUV`amsvw{|%256;CHMY^ghrs"&'27@AJ_ghptw   !"*+0267=>DEHPTafjnouv   !*+/38<HVX\`dopxy}~   !*+46AGKS[\_`kluv    % & , ; ? @ E F P R U V [ ] ` b g h q r {       " ' ( - . 2 3 6 7 E I S T X ] d h l m s t y z          # % - . 2 3 = > H I P R Y ] c g n t y }        ! * + 0 4 8 < D F N 3 : '-3Xa4:FLMV KTv{CG7=ou-3- 2 i r c!n!k"v"""##.#####$$U%Y%t%x%%%%%%%%%&&'''')))),):)=)[)_)))7*;*****+ + ++$+++,+7+++4,:,@,J,K,V,X,],o,v,w,,,-6---.:/;/q/r/////00T0U000001K1111111'2(2Z2[222222273i3t3|3333344S4T4h4i4}4~4444444)5U5`555=6H6j6l66667777 777777'7(727<7?7@7F7G7M7W7[7`7d7e7l7q7v777777777777777777777777777777778 8888%8-8.86878:8;8@8A8D8E8J8K8V8W8a8l8s8t8|8}888888888888888888888888899 9 999"9&9,9-999:9A9B9E9L9P9S9W9X9[9\9g9h9n9o9z9{9999999999999999999999:: :::::(:):0:1:9:::>:C:G:H:S:X:a:b:k::::::::::::::::::::::::::; ;;;;*;-;.;3;4;8;:;B;C;K;L;Q;S;W;X;^;_;e;f;i;q;u;;;;;;;;;;;;;;;;;;;;;;;;;; < <<<<<<<<<(<,<5<6<?<@<I<J<N<R<W<[<g<u<w<{<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<======!="=&='=+=,=0=1=9=:=?=@=I=J=S=U=`=f=j=r=z={=~===========================>>> >>>>>,>5>9>>>?>D>E>K>Z>^>_>d>e>o>s>}>>{}NOQ R E LO w { ""n#q#v#y#3$7$<$?$D$I$t$w$|$$4%6%''(())+ +R,V,--..%.*.H.M.l.q........../// /////!/"/*/+/3/4/9/;/?/@/B/C/K/L/O/P/U/V/^/_/g/h/p/r/v/w/y/z//////////////////////////////////////00 00000 0!0#0$0,0-020308090A0B0J0K0S0U0Z0[0]0^0f0g0l0m0r0s0{0|000000000000000000000000000000000001K1R1S1W1X1_1`1g1h1p1q1y1z11111111111111111111111111111111111111122 2 22222&2(2,2-2/2027282?2@2H2I2P2Q2Y2[2_2`2b2c2h2i2p2q2y2z22222222222222222222222222222222222223333333333$3%3-3.363e3t3|3333333333333333333333333333333333344 4 44444#4$4&4'4/4047484@4A4I4J4R4T4X4Y4[4\4h4i4m4n4p4q4}4~4444444444444444444444444444444444444555 555555 5(56$8%8999999p>s>~>>33333333333333333333333333333333 : O .:/;/1K193|3*55 699s>~>>>^]"cO]Br+} e RY^"? %vr(%) ~-)./YK5t5h9s?fA6E qH9M|iPtPQ4S7"UgVX8Y9[nk_ybcPbeH ik9kc;lMGpu!Gv&z|o}`w~K(aAs7+ ETf#pkJ{GXBhvA_T/lg/ZdnX:'&8.RH/M f8ZB(RVG2..../ ///"/+/:/;/@/C/L/P/V/_/h/q/r/w/z/////////////////////0 0000!0$0-03090B0K0T0U0[0^0g0m0s0|0000000000000000000011J1K1S1X1`1h1q1z1111111111111111111111122 222'2(2-20282:2@2I2Q2Z2[2`2c2i2k2q2z222222222222222222222233333%3.37383{3|33333333333333333333334 4444$4'4042484A4J4S4T4Y4\4^4`4b4d4f4h4i4n4q4s4u4w4y4{4}4~4444444444444444444445 555 5)5*55>@X;>00 0@000<@0x@UnknownGz Times New Roman5Symbol3& z Arial"qӻ&ӻ&һ&)T ,5qT ,5q!24da>a> 2QHX)?(2Monitoring of early pregnancy and diagnostics of early embrionic mortality by ultrasound and determination of PAG and progesterone in cows nprvanovic nprvanovic  F!Microsoft Office Wordov dokument MSWordDocWord.Document.89q