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Diseminacija programske podrške zračnim prijenosom je metoda nadograđivanja zastarjele ili neispravne programske podrške na više uređaja odjednom s udaljene lokacije. Over-The-Air firmware update is the process of upgrading firmware of multiple devices at once from a remote location. .

U industriji Interneta stvari se dovodi puno novih tehnologija i tehnoloških rješenja na tržište kako bi se zadovoljile potrebe ostvarivanja komunikacije s krajnjim uređajima. LoRa tehnologiju bežične komunikacije je razvila tvrtka Semtech. Dizajnirana je tako da omogućuje dalekometnu komunikaciju između krajnjih uređaja i pristupnika uz malu potrošnju energije. U ovom radu je predstavljen sustav za ispitivanje propusnosti komunikacijskoga kanala izvedenog u LoRa tehnologiji. Sustav za mjerenje propusnosti komunikacijskog kanala se izvodi u fizičkom sloju LoRa-e i namijenjen je mjerenju propusnosti u mreži s N uređaja ovisno o parametrima LoRa komunikacije. Kao krajnji uređaj ili čvor se koristi LoPy mikrokontroler. Ulogu mrežnog pristupnika obnaša Raspberry Pi 3B+ računalo sa spojenim InAir9 modulom. In the Internet of Things industry there are many new emerging technologies and solutions which are used to establish communication with end devices. LoRa wireless communications technology was developed by Semtech. It was designed to enable long range transmissions between end-devices and gateways with low energy consumption. A system to measure throughput in LoRa communications channel is presented in this paper. The system is based on the LoRa physical layer and is intended for measurements in a network with N number of nodes with different parameter settings. LoPy microcontrollers are used as end devices and the gateway is implemented using Raspberry Pi 3B+ with connected InAir9 module.

This Thesis investigates intraday price observations for six European emerging stock markets and explores the effectiveness of range-based volatility estimators with an empirical analysis. The theory of Realized Volatility utilizes intraday data to estimate the integrated volatility. However, Realized Volatility is often biased due to microstructure noise. The Two Times Scale Estimator is an unbiased estimator of the integrated volatility. For each of the indices a consistent and asymptotically unbiased estimator of the integrated volatility is determined using intraday price observations and the Two Times Scale Estimator. As intraday price observations provide additional insight in the price changes during trading hours, it also has limitations in industry wide applicability as intraday data is not always available. The reasons can vary, from restricted data to illiquid markets. As a reasonable alternative for many applications in finance this Thesis suggest the use of range-based volatility estimators that utilize only a limited number of intraday price observations, i.e. the Open, High, Low and Close price observations. The standard range-based volatility estimators are extended with the information captured in overnight jumps and contribute to the already rich literature on financial volatility. The second part of this Thesis focuses on the challenge of ranking the results. As far as we are aware the literature had not been unanimous on the ranking methodology. This research compares several ranking methodologies and attempts to provide more guidelines in the choice of the ranking methodology given its purpose. The existing ranking methodologies (loss functions, coefficient of efficiency and the Mincer Zarnowitz regression) focus on an overall fit, while the extreme movements are often insufficiently covered. This research employs the upper tail dependence coefficient, a result of the Gumbel copula function, for comparison purposes when the focus of interest are the tails of the distribution. The upper tail dependence is a complementary ranking methodology to the standard loss functions or coefficient of efficiency approach. The results show that range-based volatility models are appropriate alternatives to the Two Times Scale Estimator and that the neither the standard deviation nor the daily squared return have been selected in any of the ranking methodologies. Ova disertacija istražuje opažanja intradnevnih cijena šest izranjajućih europskih dioničkih tržišta te empirijskom analizom istražuje učinkovitost rasponskih procjenitelja volatilnosti. Teorija realizirane volatilnosti koristi intradnevne podatke za procjenu integrirane volatilnosti. Međutim, realizirana volatilnost je često pristrana zbog raznih mikrostrukturnih efekata. Glavni procjenitelj u ovom radu je nepristrani procjenitelj integrirane volatilnosti koji procjenjuje volatilnost u dvije „faze“, te se razlikuje od realizirane volatilnosti. U radu se koristi skraćenica engleskog naziva „Two Times Scale Estimator“, odnosno TTSE, kako bi se označio nepristrani procjenitelj integrirane volatilnosti. Za svaki od šest burzovnih indeksa određen je odgovarajuć i asimptotski nepristran procjenitelj integrirane volatilnosti koristeći TTSE procjenitelj na bazi tržišnih cijena promatranih visokom intradnevnom frekvencijom. Bitno je naglasiti da sama opažanja intradnevnih cijena pružaju dodatan i veoma vrijedan uvid u cjenovne promjene tijekom sati trgovanja na burzi. Međutim, postoje određena ograničenja kod široke primjene u cijeloj financijskoj industriji budući da intradnevni podaci nisu uvijek dostupni za svako tržište odnosno za svaki financijski instrument. Razlozi potonjeg variraju od ograničenih podataka do nelikvidnih tržišta. Kao razumnu alternativu za razne primjene u području financija, ova disertacija sugerira korištenje rasponske procjenitelje volatilnosti koji koriste samo ograničen broj opažanja intradnevnih cijena, tj. cijena otvaranja, najviša cijena, najniža cijena i cijena zatvaranja tijekom dana trgovanja. U radu se koristi skraćenica engleskog naziva „open, high, low, close” (OHLC). Standardni rasponski procjenitelji volatilnosti ne uzimaju u obzir informaciju koja se promatra van standardnih sati trgovanja. Promjene u cijenama koje se realiziraju van standardnih sati trovanja nazivaju se „prekonoćnim skokovima“. Prekonoćni skokovi u cijenama promatraju se kao razlika između početne cijena trenutnog dana i završne cijene prethodnog dana. U radu su standardni rasponski procjenitelji obogaćeni prekonoćnim skokovima te s ovim rad doprinosi već bogatoj literaturi o procjeniteljima financijske volatilnosti. Drugi dio ovog rada usredotočen je na izazov rangiranja rezultata procjenitelja volatilnosti. Koliko je autoru ovog rada poznato, literatura nije bila jednoglasna u metodologiji rangiranja ovih procjenitelja, te smatra da metoda rangiranja uvelike utječe na konačan optimalan izbor procjenitelja. Ovo vii istraživanje uspoređuje nekoliko metodologija rangiranja te nastoji ponuditi smjernice u izboru metodologije rangiranja s obzirom na svoju svrhu. Postojeće metodologije rangiranja (kao što su funkcija gubitka, koeficijent učinkovitosti i Mincer Zarnowitzeva regresija) usredotočene su na cjelokupnu statističku raspodjelu, dok su ekstremne promjene u volatilnosti cijena često nedovoljno pokrivene. Ovo istraživanje koristi koeficijent gornje ovisnosti repa (eng. „upper tail-dependence“), koji je rezultat Gumbelove Copula funkcije, za svrhe usporedbe kada su fokus interesa upravo sami repovi raspodjele. Zavisnost u gornjem dijelu repa raspodjele smatra se komplementarnom metodologijom rangiranja koja uz standardnu funkciju gubitka ili uz pristup koeficijenta učinkovitosti, upotpunjava analizu rangiranja procjenitelja volatilnosti. Rezultati pokazuju da su modeli volatilnosti na bazi raspona prikladne alternative procjenitelju TTSE i da ni standardna devijacija ni dnevni kvadratni prinos, koji se među standardnim rasponskim procjeniteljima volatilnost smatraju najpopularnijim, nisu odabrani u bilo kojoj metodologiji rangiranja. Među izabranim rasponskim procjeniteljima volatilnosti su Parkinson, Garman-Klass, High-Low, Roger-Satchell te Yang-Zhang. Optimalan model rasponske volatilnosti ovisi o primjenjenoj metodi rangiranja, odnosno o samoj primjeni u praksi.

The aim of the present study was to investigate the effect of follicle size from which oocytes originate on their subsequent ability to be fertilized and to undergo early embryonic development in vitro. Cumulus oocytes complexes (COCs) aspirated from small (≤ 5 mm) and large (> 5 mm) follicles of abattoir-derived ovaries were graded for their morphological appearance and were cultured to assess their developmental competence. In order to study the kinetics of early cleavage, the number of cleaved embryos was recorded at 24, 27, 30, 33, 36 and 48 hours post insemination (hpi). A rapid rise in the rate of cleaved embryos was observed by 27 hpi, for both oocytes collected from ≤5 mm and >5 mm follicles. However, oocytes recovered from follicles >5 mm cleaved in significantly higher proportion at 24 to 30 hpi, which was reflected to higher overall cleavage rate at 48 hpi. The kinetics of early cleavage was consistent with subsequent development of embryos, that is, the oocytes from >5 mm follicles which completed the first cell division faster developed to morula stage at Day 5 and blastocyst stage at Days 6 and 7 in higher proportion than oocytes from ≤5 mm follicles. Hatching rate on Day 9 was significantly higher when oocytes originate from follicles > 5 mm. Total cell number was not affected by follicle size. The results showed that oocytes derived from follicles > 5 mm display higher developmental competence than oocytes from ≤5 mm follicles, in terms of timing of first cleavage, timing of blastocyst development and overall blastocyst rate. The selection of oocytes based on follicle size and kinetics of cleavage could be useful tools in selecting the best embryos for transfer.

Modern Portfolio Theory (MPT) according to Markowitz states that investors form mean-variance efficient portfolios which maximizes their utility. Markowitz proposed the standard deviation as a simple measure for portfolio risk and the lower semi-variance as the only risk measure of interest to rational investors. This paper uses a third volatility estimator based on intraday data and compares three efficient frontiers on the Croatian Stock Market. The results show that range-based volatility estimator outperforms both mean-variance and lower semi-variance model., {"references":["Aljinović, Z., Marasović, B., Šego, B., Financijsko modeliranje, II\nizmijenjeno i dopunjeno izdanje, Ekonomski fakultet Split, 2011, pp.\n121-153.","Aljinović, Z., Marasović, B., Vidović, J., \"The alternative risk measures\nin Excel\", MIPRO 2010, Proceedings of the 33rd International\nConvention: Computers in Education, Opatija, Croatia, 2010, pp. 152-\n157.","Andersen, T.G., Bollerslev, T., Diebold, F.X., and Labys, P., \"The\ndistribution of realized exchange rate volatility\", Journal of the\nAmerican Statistical Association 96, 2001, pp. 42-55.","Arnerić, J. and Matković, M., \"Ranking Open, High, Low, Closing\nVolatility Estimators\", Split Faculty of Economics - Working paper,\n2013.","Bawa, S. and Vijay S., \"Optimal Rules For Ordering Uncertain\nProspects\", Journal of Financial Economics, v2(1), 1975, pp. 95-121.","Cheng, P. and Woverton, M., \"MPT and the Downside Risk Framework:\nA Comment on Two Recent Studies\", Journal of Real Estate Portfolio\nManagement, vol 07, no.2, 2001.","Cont, R., \" Empirical properties of asset returns: stylized facts and\nstatistical issues\", Journal of Quantitative Finance, vol. 01, 2001, pp.\n223-236.","Dacarogna, M.M., Muller, U.A., Olsen, R.B, and Pictet, O.V.,\n\"Modelling short term volatility with GARCH and HARCH models\",\npublished in \"Nonlinear Modelling of High frequency Financial Time\nSeries.\", edited by Christian Dunis and Bin Zhou, John Wiley,\nChichester, 1998, pp. 161-176.","Duque, J., and Paxson, D., \"Empirical Evidence on Volatility\nEstimators\", Working Paper, Universidade Tecnica de Lisboa and\nUniversity of Manchester, 1997.\n[10] Foo, T., Eng, S., \"Asset Allocation in a Downside Risk Framework\",\nJournal or Real Estate Portfolio Management, Vol.06, No.3, 2009.\n[11] Garman, M.B., and Klass, M.J., \"On the Estimation of Security Price\nVolatilities from Historical Data\", Journal of Business, 53, 1980, pp. 67-\n78.\n[12] Harris, R.D.F., Yilmaz, F. \"Estimation of the Conditional Variance-\nCovariance Matrix of Returns using the Intraday Range\", University of\nExerer, XFi Centre for Finance and Investment, Working paper: 07/11,\nOctober, 2007\n[13] Konno, H., Waki, H., Yuuki, A., \"Portfolio Optimization under Lower\nPartial Risk Measures\", Asia-Pacific Financial Markets, vol.9,, 2002,\npp. 127-140, Kluwer.\n[14] Mao, James C. T., \"Models Of Capital Budgeting, E-V Vs. E-S\" Journal\nof Financial and Quantitative Analysis, v5(5), 1970, pp. 657-676.\n[15] Markowitz, H., \"Portfolio Selection: Efficient Diversification of\nInvestments\", Journal of Finance, Vol.7, No.1, 1952, pp. 77-91.\n[16] Markowitz, H., \"Portfolio Selection: Efficient Diversification of\nInvestments\", John Wiley and Sons, 1959.\n[17] Parkinson, M., \"The Extreme Value Method for Estimating the Variance\nof the Rate of Return\", Journal of Business, 53, 1980, pp. 61-65.\n[18] Rogers, L.C.G., and Satchell, S.E., \"Estimating Variance from High,\nLow, and Closing Prices\", Annals of Applied Probability 1, 1991, pp.\n50-512.\n[19] Sing, T. F., Ong, S. E., \"Asset Allocation in a Downside Risk\nFramework\", Journal of Real Estate Portfolio Management, Vol.6, no.3,\n2000, pp. 213–224.\n[20] Sivitanides, P. S., \"A Downside-risk Approach to Real Estate Portfolio\nStructuring\", Journal of Real Estate Portfolio Management, Vol.4, no.2,\n1998, pp. 159–168."]}

U radu je prikazan prijedlog strategije brendiranja ruralnog turizma Zadarske županije na temelju primarnog istraživanja - dubinskog intervjua s direktorima Turističkih zajednica Zadarske županije. Kao posljedica usmjerenosti hrvatskog turizma na maritimne prostore, ruralni turizam i turizam na seljačkim gospodarstvima u RH je u početnoj fazi. Najveći iskorak u turističkoj valorizaciji ruralnih prostora učinile su Istarska i Dubrovačko- Neretvanska županija, kojima prema prirodnim, kulturnim, povijesnim i gastronomskim posebnostima Zadarska županija konkurira, međutim prema stupnju razvoja ruralnog turizma za njima uvelike zaostaje. Kako globalna turistička djelatnost postaje sve konkurentnija, mnoge turističke destinacije svoju priliku vide u brendiranju. Brendiranjem se kod potrošača stvara prepoznatljivost turističke destinacije, kao i „emotivna veza“ između turističkih korisnika i same destinacije. Kao rezultat, turistička destinacija bilježi bolju posjećenost, veću potražnju za proizvodima i uslugama, porast cijena nekretnina, poboljšanje turističkih rezultata te u konačnici zadovoljstvo lokalnog stanovništva. U radu se također prikazuje osvrt na Europske smjernice razvoja ruralnog turizma, odnosno Direktive Europske unije zacrtane posebnim dokumentom koji daje preporuke za strateške pravce razvoja turističke destinacije, te se ocjenjuje dostignuta razina razvoja ruralnog turizma u RH sukladno navedenim Direktivama, na primjeru Zadarske županije. U tijeku pisanja rada provedeno je istraživanje koje je potvrdilo da ruralni turizam Zadarske županije ne iskorištava u potpunosti svoj potencijal, što se može promijeniti kroz razvoj brenda koji će omogućiti prepoznatljivost, povećanje kvalitete i raznolikosti turističke ponude te društveni i gospodarski napredak, kako Zadarske županije, tako i cijele regije.

The application of assisted reproductive technology (ART) in stockbreeding has tremendously changed the genetic improvement rates in breeding programs and strategies. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. Ultrasound-guided ovum pick-up (OPU) followed by in vitro embryo production (IVP) offers an alternative to produce offspring from genetically superior dams, pregnant cows and heifers, post-partum cows and even from prepubertal heifers and cows with reproductive disorders. Over the last decades basic and applied research projects have been conducted in the Croatia with the aim of application and commercialization of ART in our cattle breeding schemes. The Laboratory for Assisted Reproduction at the Faculty of Veterinary Medicine, University in Zagreb, along with the Center for reproduction and animal breeding of Croatia, represent the nucleus unit for application of these modern embryo technologies in our stock breeding schemes. During a 10 years period (2002 to 2012) the embryo transfer team of the Center for reproduction and animal breeding of Croatia transferred 368 cattle embryos and produced 166 calves (75 bulls, 89 heifers and 2 recipients had abortion). The same team has also flushed Holstein-Friesian (n=45) and Simmental (n=87) superovulated donors. On average the ovulation rate (the number of corpora lutea), oocyte rate and embryo rate were: 409 CL, (X=9.8), 225 oocytes (X=5.0) and 98 transferrable embryos (X=2.2) in Holstein-Friesian donors and 1310 CL, (X=15.1), 1271 oocytes (X=14.6) and 794 transferrable embryos (X=9.2) in Simmental donors. The number of ovulated follicles after superovulation, the numer of produced oocytes and transferrable embryos was significantly higher for Simmental comparing to Holstein-Friesian donor cows. In vitro embryo production (IVP) is a newer and more flexible approach to produce cattle embryos, although it is technically more demanding and requires specific and costly laboratory equipment. A present standard protocol for IVP of bovine embryos in the Laboratory for Assisted Reproduction at the Faculty of Veterinary Medicine, University in Zagreb is based on the use of BoviPure® for sperm preparation for in vitro fertilization (IVF) and synthetic oviductal fluid with amino acids and bovine serum albumin (SOFaaBSA) for in vitro culture (IVC). During the 10 years period more than 230 OPU sessions were carried out in 63 stimulated and/or cyclic heifers and cows, resulted with 578 transferable embryos. The animals submitted to ART are under strict sanitary control what ensure the minimum risk for transmition of pathogenic agents. Considering all this facts, not only that we facilitate genetic improvement in the cattle industry by obtaining a large number of calves from cattle of high genetic quality, but we also have the opportunity to control health of the animals and indirectly of humans by producing the final product free from infectious diseases.

The aim of the present study was to compare the efficiency Ovum Pick-Up (OPU) and subsequent blastocyst development of oocytes collected from three cattle breeds. OPU was performed in 14 nonlactating donor cows (4 Simmental (S), 5 Charolais (CH) and 5 Holstein-Friesian (HF)). Cows were synchronized with PGF2 and stimulated with pFSH, twice a day during two days (Folltropin®, Bioniche, a total dose: 200 mg NIH-FSH-P1). Four OPU sessions were performed per cow. The number of aspirated follicles, the number of retrieved oocytes and oocyte recovery rate were recorded. The recovered cumulus oocyte complexes (COCs) were morphologically classified into four (1-4) quality categories. Grade 1 and 2 oocytes were matured, fertilized and cultured in vitro for 9 days. Embryo yield was recorded at day 5, 6, 7 and 9. The number of retrieved COCs per OPU session was significantly higher for Simmental and Charolais donor cows comparing to Holstein-Friesian cows (9, 5, 8, 9 and 4, 6 respectively), due to a higher number of follicles present in the Simmental and Charolais ovaries. In terms of blastocyst rates at days 5, 6, 7 and 9 of in vitro culture, no statistical differences were observed between breeds. In conclusion, we reported that Simmental and Charolais donor cows showed significantly better results in number of follicles punctured, total number of oocytes and number of grade 1 and 2 oocytes recovered by OPU, but there were no differences in developmental competence of retrieved oocytes of similar quality between three donor breeds.

U radu je prikazan prijedlog strategije brendiranja ruralnog turizma Zadarske županije na temelju intervjua s direktorima Turističkih zajednica Zadarske županije. Kao posljedica usmjerenosti hrvatskog turizma na maritimne prostore ruralni turizam i turizam na seljačkim gospodarstvima je u početnoj fazi. Najdalji iskorak s valorizacijom ruralnih prostora su učinile Istarska i Dubrovačko-Neretvanska županija, kojima prema prirodnim, kulturnim, povijesnim i gastronomskim posebnostima Zadarska županija konkurira, međutim prema stupnju razvoja ruralnog turizma za njima uvelike zaostaje. Kako globalna turistička djelatnost postaje sve konkurentnija, mnoge turističke destinacije svoju priliku vide u brendiranju. Brendiranjem kod potrošača stvaramo jednu vrstu emocija prema brendiranoj destinaciji, što za rezultat ima bolju posjećenost, veću potražnju za proizvodima i uslugama, porast cijena nekretnina, poboljšanje turističkih rezultata te u konačnici zadovoljstvo lokalnog stanovništva.

Ultrasound-guided ovum pick-up (OPU) followed by in vitro embryo production offers an alternative to produce offspring from genetically superior dams, pregnant cows and heifers, post-partum cows and even from prepubertal heifers and cows with reproductive disorders. Over the last decade basic and applied research projects have been conducted in the Laboratory for Assisted Reproduction of Faculty of Veterinary Medicine in Zagreb. A present standard protocol for IVP of bovine embryos is based on the use of BoviPure® for sperm preparation for in vitro fertilization (IVF) and synthetic oviductal fluid with amino acids and bovine serume albumine (SOFaaBSA) for in vitro culture (IVC). During the 10 years period more than 230 OPU sessions were carried out in 63 stimulated and/or cyclic heifers and cows, resulted with 578 transferable embryos. Different synchronization treatments for OPU/IVP donor heifers were investigated: prostaglandin F2 administration without or with dominant follicle removal (DFR) and norgestomet ear implant insertion with DFR. The OPU/IVF results were compared in PMSG and pFSH stimulated donors with or without DFR, as well in cyclic donors when OPU was performed twice a week during a month period. Follicular dynamics and in vitro embryo production after repeated superovulation with pFSH in Simmental heifers were monitored, as well the effect of the follicle size on developmental competence of bovine oocytes. The effect of the origin of the immature oocytes (slaughterhouse or OPU), a well the effect of donor breed (Charolais, Simmental and Holstein.Frieian) for OPU/IVP were compared. Transvaginal OPU/IVP was also performed in problem cows, suffering from cystic ovaries or post-partum anestria due to endometritis with satisfactory results. Using OPU/IVP good quality bovine blastocysts were produced in vitro suitable for embryo transfer or cryopreservation, comparable to other laboratories for IVP of bovine embryos.

The objective of this study was to investigate the effect of follicle size on developmental competence of oocytes collected by Ovum pick up (OPU). Immature cumulus-oocyte-complexes (COCs) were obtained by aspirating follicles ≤ 5 mm or > 5 mm. Transvaginal ultrasound guided oocyte collection was performed in 4 Charolais donor cows. Cows were synchronized with PGF2 and stimulated with pFSH, twice a day during two days (Folltropin®, Bioniche). OPU was performed 24 hours after the last FSH injection. Total of 6 OPU sessions were performed on each cow. The number of aspirated follicles, retrieved oocytes and oocyte recovery rate (RR) were recorded. The recovered oocytes were morphologically classified into four (1-4) quality categories. Grade 1 and 2 oocytes were matured, fertilized and subsequently cultured in vitro for 9 days. In order to study the kinetics of early cleavage, the number of cleaved embryos was recorded at 24, 27, 30, 33, 36 and 48 hours post insemination (hpi). Embryo yield was recorded on day 5, 6, 7 and 9 of culture in vitro. Data were analyzed by t- test and Wilcoxon test. There was no difference in recovery rate or quality of oocytes collected from large or small follicles. Oocytes originate from follicles > 5 mm cleaved in significantly higher proportion 24 hpi (p 5 mm reached the morula stage compared to 13, 1% of morulas when oocytes originated from follicles ≤ 5 mm (p 5 mm (48, 1% vs. 40, 2%), although without statistical difference. Hatching rate on Day 9 was significantly higher when oocytes originate from follicles > 5 mm (29, 7% vs. 10, 7%, p

The aim of the study was to compare the effect of different synchronization protocols on oocyte retrieval and blastocyst production in vitro. Twelve Simmental heifers were randomly allocated to one of three synchronization groups. In the 1st group heifers were synchronized with two PGF2α injections in 11-days interval. In the 2nd group heifers were synchronized the same way and dominant follicles (DF) were removed 36 hours before pFSH stimulation. In the 3rd group a progestin ear implant was inserted for 10 days and DF were aspirated 36 hours before pFSH stimulation. The same stimulation protocol with pFSH, starting on Day 9/10 of cycle, was used in all treatment groups. Ovum pick-up (OPU) was performed 48 hours after the last pFSH injection. The number of aspirated follicles, number and quality of retrieved oocytes were recorded. Oocytes were matured, fertilized and cultured in vitro in SOFaaBSA until Day 9. The mean DF diameter and the mean number of expanded oocytes showed a tendency towards signififi cance (0.05 ≤ P ≤ 0.10) in the progestin treatment group. No signififi cant differences between treatment groups were observed in terms of cleavage rate on Day 2, morula/blastocyst rate on Day 7 and hatched blastocysts rate on Day 9. The results indicated that progestin treatment did not prevent the establishment of follicular dominance on the ovaries of heifers. Furthermore, the synchronization protocol did not affect the number of follicles and the recovered oocytes, or the number of in vitro produced embryos.

The aim of this research was to compare the developmental competence of oocytes recovered from slaughterhouse ovaries (SO) and those recovered in vivo by ovum pick up (OPU). OPU was performed in 6 donor heifers synchronized with PGF2 and stimulated with pFSH, twice a day during two days. OPU was repeated once a week for six consecutive weeks. The control group consisted of slaughterhouse-derived oocytes. The number of aspirated follicles, retrieved oocytes and oocyte recovery rate were recorded. Oocytes of grade 1 and 2 were matured, fertilized and cultured in vitro in SOFaaBSA until Day 9. The cleavage rates on Day 2, the total number of morulas and blastocysts on Day 7 and the numbers of hatched blastocysts on Day 9 were recorded. Differential staining of the inner cell mass (ICM) and trophectoderm cells (TE) were performed on randome sample of Day 7 blastocysts (n=24). Significantly more follicles were aspirated from SO (19.3 versus 11.5, P

Uspješnost zajedničkog INA-MOL projekta na području Dravica (Hrvatska) - Zalata (Mađarska), možemo zahvaliti primjeni novog istraživačkog koncepta. Upotrebom u svijetu provjerene metode – sekvencijske stratigrafije – otvorena su vrata novom istražnom projektu Novi Gradac (Hrvatska) – Potony (Mađarska).

Cilj našeg istraživanja bio je istražiti utjecaj faze spolnog ciklusa i promjera folikula na razvojnu sposobnost polučenih jajnih stanica u postupku dozrijevanja oplodnje in vitro. Iz 70 jajnika zaklanih krava simentaske pasmine aspirirali smo ukupno 1.682 folikula promjera do 8 mm tijekom ljetnog razdoblja (lipanj i srpanj). Na osnovi morfološke ocjene, parove jajnika smo razvrstali u 3 različite faze spolnog ciklusa, a folikule u dvije kategorije (> od 3 mm i < od 3 mm). Pregledom aspirirane tekućine pronašli smo 737 jajnih stanica, od kojih 475 u kategoriji manjih od 3 mm, a 262 u kategoriji većih od 3 mm. Uspjeh aspiracije iznosio je 43, 8%. Dobivene nezrele jajne stanice morfološki smo ocjenili i svrstali u 3 kategorije, od kojih su u postupak dozrijevanja ušle samo jajne stanice I i II kategorije („ dobre“ jajne stanice). Odabrane jajne stanice stavili smo u medij za dozrijevanje te ih nakon inkubacije od 24 sata oplodili duboko smrznutom spermom bika pripremljenoj na gradijentu Percoll® ; . Drugog dana nakon oplodnje utvrdili smo postotak brazdanja od 50, 6%. Dobivene zametke smo uzgajali u definiranom mediju SOFaaBSA do stadija morula i blastocista. Nakon 7. dana uzgoja dobili smo 59 (15, 8%) morula i blastocista, a 11. dana 37 (9, 9%) blastocista i 22 (5, 9%) izlijeganih blastocista koje smo klasificirali po IETS standardima. Nismo ustanovili značajnu razliku u sposobnosti uspjeha dozrijevanja, oplodnje i uzgoja jajnih stanica među različitim fazama spolnog ciklusa niti među kategorijama folikula. Budući da su Makek i sur. (2000.) u proljetnom razdoblju, uz iste uvjete uzgoja te po istom protokolu rada u istom laboratoriju, dobili veći broj trasferbilinih zametaka, možemo pretpostaviti da je na dobivene rezultate utjecala sezona (ljeto) u kojoj su provedena istraživanja.

The topic of our interest in this paper is the application of Electronic Technology in the hotel industry Business. Reserving hotel accommodation can be a lengthy and frustrating process for potential customers. Business is simplified with the development of Electronic and Internet Business and hotel services are easily available to customers regardless of their location. The paper looks into the importance of the global distribution network for the hotel industry. Modern Business methods contribute towards maximizing the revenues realized per hotel room.

The purpose of this study was to evaluate weather the addition of cysteamine during in vitro culture (IVC) of bovine embryos enhances embryo development. Cumulus-oocytes complexes collected from ovaries of slaughtered donors were matured in vitro in TCM 199 supplemented with 10% FCS, FSH/LH (Pergonal, 75/75 IU, Serono), 1 µ ; g/ml estradiol-17ß and 100 µ ; M cysteamine for 24 hours. After IVM the oocytes were fertilized (Day 0). Following fertilization embryos were cultured in SOFaaBSA without glucose for 48 h. Day 3 embryos were transfered to SOFaaBSA with 1, 5 mM glucose (SOF g) supplemented with 0 or 100 µ ; M cysteamine and cultured for 48 h. After this, embryos were transferred in SOF g without cysteamine and cultured until Day 9. Total of 474 oocytes were used in five repetitions. The endpoints for evaluation were: Day 2 cleavage rate ; Day 7 blastocyst rate and Day 9 hatching rate. The percentage of embryos that developed to the blastocyst stage on Day 7 was significantly higher (p

The success of ovum pick-up (OPU) and in vitro embryo production considerably depends on the number and the quality of retrieved oocytes per session and also on the culture conditions. The aim of the study was to compare the effect of different synchronization protocols on oocyte retrieval and blastocyst production in vitro in FSH stimulated Simmental heifers. Twenty-eight normal cyclic Simmental heifers, aged between 14 and 20 months were selected based on transrectal palpation for oocyte donors. They were randomly allocated to one of three synchronization groups. In the 1st (PGFx2) group heifers (n=10) were synchronized with two prostaglandin F2 injections at 11-day interval. In the 2nd (PGF + DFR) group heifers (n=8) were synchronized with two prostaglandin F2 treatments at 11-day interval and dominant follicles (>8 mm) were aspirated (DFR: dominant follicle removal) 36 hours before FSH stimulation. In the 3rd (Crestar + DFR) group (n=10) a norgestomet ear implant without the prescribed oestrogen injection (Crestar® ; , Intervet, Begium) was inserted for 10 days. The implants were removed and dominant follicles were aspirated 36 hours before FSH stimulation. The stimulation with pFSH (Folltropin® ; -V, Vetrepharm Inc., London, Canada) was the same fir all three groups and it was administered twice a day, during two days beginning on the day 9 or 10 of the estrous cycle. The total amount of pFSH administered was 200 mg NIH-FSH-P1 in 4 equivalent doses. OPU was performed 48 hours after the last FSH injection. Only good quality oocytes were matured, fertilized and cultured in vitro in SOFaaBSA till the Day 9. Cultured embryos were evaluated morphologically according to IETS standards. The cleavage rates on Day 2, the total number of morulas (M) and blastocysts (Bl) on Day 7 and the number of hatched blastocysts (hBl) on Day 9 were recorded. The results were statistically analyzed with ANOVA (StatSoft, Statistic, version 7.1.) and with Tukey, s tests post-hoc analysis.The results are presented in the table. Table: Effect of different synchronization protocols on oocyte retrieval and IVF/IVC results in FSH stimulated Simmental heifers (mean± ; SD) Synchronization protocol Follicles aspirated Oocytes retrieved Oocytes in IVP Cleavage (%) M/Bl (%) hBL (%) PGFx2 group (n=10) 14.5 (± ; 3.9)a 10.3 (± ; 5.2)b 9.5 (± ; 5.6)a 86.8 (± ; 1.8)a 41.1 (± ; 2, 2) a 22.4 (± ; 1.8) a PGF + DFR group (n=8) 13.3 (± ; 3.9) a 7.6 (± ; 4.0) a 6.9 (± ; 4.5) ab 91.0 (± ; 1.3) a 28.9 (± ; 2.2) b 12.4 (± ; 1.4) b Crestar + DFR group (n=10) 13.1 (± ; 4.1) a 9.1 (± ; 4.1) ab 5.9 (± ; 3.6) b 94.7 (± ; 0.8) a 43.6 (± ; 2.0)a 23.2 (± ; 1.5) a Values with different superscripts within the same columns differ significantly (P

Protection of the autochthonous breeds in Croatia is performing more than one decade. National protection program is based on government subvention, and negative trend in endangered breeds was stopped, thanks to them. During the years of protection programs conduction none of autochthonous breeds were lost. Protection programs of autochthonous breeds are conduced primary through in situ models. Some new opinion concerning protection of autochthonous breeds point out their economic utilization, integration in protected areas, collecting genetic materials and storage in national “ Genebank” . Croatia has lot of protected areas, fullness of pasture land in which autochthonous breeds can be easily integrated. It is necessary to establish national ‘ Genebank’ , and new knowledge and instructions about storage genetic materials open the possibility of its quickly foundation and occupancy. Collecting somatic cells of autochthonous, primary endangered breed, is solution, mainly because of its simplicity of collecting and storage procedure, and lowest investment assets at the beginning. Along with collecting tissue cells, collecting of semen, embryos and oocytes is necessary. Information about tissue samples stored in national gene bank will be integrated in national animal information system.

Numerous researches suggest that the intrinsic quality of the oocyte is the critical factor determining the proportion of the oocytes developing to the blastocyst stage, although culture conditions also affect developmental potential of the embryo. The aim of this research was to compare the embryo developmental competence of immature oocytes recovered from slaughterhouse ovaries (SO) and those recovered in vivo by ovum pick up (OPU). Transvaginal ultrasoung guided (Pie Medical, Netherlands) oocyte collection was performed in 6 Simmental donor heifers. Heifers were synchronized with PGF2 and stimulated with pFSH, twice a day during two days (Folltropin® ; ; -V, Vetrepharm Inc., London, Canada, a total dose: 200 mg NIH-FSH-P1). OPU was performed 48 hours after the last FSH injection and procedure was repeated once a week for six consecutive weeks. Oocytes were matured, fertilized and cultured in vitro in SOFaaBSA until Day 9. The cleavage rates on Day 2, the total number of morulas (M) and blastocysts (Bl) on Day 7 and the numbers of hatched blastocysts (hBl) on Day 9 were recorded. Differential staining of the inner cell mass (ICM) and trophectoderm cells were performed on random samples on Day 7 blastocysts (n=24). The results were analyzed with ANOVA (StatSoft, Statistic, version 7.1.) and with Tukey’ s tests post-hoc analysis. The results are presented in the table. Table: Effect of bovine oocyte origin on in vitro development of embryos (mean± ; ; SD) Oocyte origin N Cleavage (%) M/Bl (%) HBL (%) Slaughterhouse oocytes 398 76.83 (± ; ; 2.31)a 29.96 (± ; ; 2.08) a 16.78 (± ; ; 0.78) a OPU-derived oocytes 212 81.29 (± ; ; 8.83) a 44.71 (± ; ; 7.20)b 35.57 (± ; ; 5.87) b Values with different superscripts within the same columns differ significantly (P

The present study examined the effect of cysteamine during in vitro maturation (IVM) of bovine oocytes on blastocyst development and embryo quality. After maturation in control (0µ M) or cysteamine supplemented medium (100µ M) oocytes were fertilized and cultured for 9 days. The endpoints for evaluation were: Day 2 cleavage rate ; Day 7 blastocyst rate ; Day 9 hatching rate, total cell number and differentiation of the embryonic cells into inner cell mass cells (ICM) and trophectoderm cells (TE) in Day 9 embryos. No beneficial effect regarding to maturation or cleavage rate was related to the addition of cysteamine. However, the addition of cysteamine resulted in significantly higher percentage of morulas and blastocysts on Day 7 (p

The objective of this study was to evaluate the effect of cysteamine addition to the in vitro culture on bovine embryo quality. Bovine oocytes collected from ovaries of slaughtered donors were matured in vitro in medium 199 supplemented with 10% FCS, FSH/LH (Pergonal, 75/75 IU, Serono), 1 µ ; ; g/ml estradiol-17ß and 100 µ ; ; M cysteamine. Matured oocytes were fertilized with modified Tyrode's solution supplemented with 10 µ ; ; g/ml heparine, 0, 5 µ ; ; g/ml hypotaurine, 0, 5 µ ; ; g/ml epinephrine and 6 mg/ml BSA. Fertilization in vitro was performed with frozen/thawed bull semen prepared using BoviPure® ; ; separation protocol. Following fertilization embryos were cultured in SOFaaBSA without glucose. After 48 h embryos were transfered to SOFaaBSA with 1, 5 mM glucose (SOF g) with or without cysteamine (Cys). Embryos that were developed to the ≥ 4 cell stage after 48 h of culture were alocated in four different groups: 1) 0 Cys ; 2) 50 µ ; ; ; M Cys ; 3) 100 µ ; ; ; M Cys and 4) 200 µ ; ; ; M. Cys. After 48 h of IVC in those media the embryos were transfered in SOF g without cysteamine and cultured until Day 9. Total of 958 oocytes were used in seven repetitions. The number of cleaved embryos on Day 2, the total number of morulas (M) and blastocysts (Bl) on Day 7 and the numbers of hatched blastocysts (hBl) on Day 9 were registered. Differential staining of the inner cell mass (ICM) and trophectoderm (TE) cells were performed on Day 9 blastocysts. The average values of the IVF results were: 83% cleaved on Day 2, 26, 2% of M+Bl on Day 7 and 11, 3% of hBl on Day 9 of development, without significant differences between treatments (p>0.05), although the differences in the percentage of morulas and blastocysts on Day 7 between 0 Cys group and 100 µ ; ; ; M Cys group were over 10% (21.9% vs 33.3%). Number of total and inner cell mass cells (mean± ; ; ; SEM) in Day 9 Bl Group N total cell no. ICM cell no. Proportion (%) 0 Cys 11 125.9± ; ; ; 11.5 a 33.4± ; ; ; 4.1b 26.6± ; ; ; 1.6b 50µ ; ; ; M Cys 12 129.9± ; ; ; 10.9 a 37.9± ; ; ; 3.9 ab 29.6± ; ; ; 1.5ab 100µ ; ; ; M Cys 15 141.6± ; ; ; 9.8a 48.7± ; ; ; 3.6a 33.7± ; ; ; 1.3a 200µ ; ; ; M Cys 7 85.0± ; ; ; 14.4b 22.6± ; ; ; 5.2b 26.5± ; ; ; 1.9b Values with different superscripts within the same columns differ significantly (P

In der vorliegenden Untersuchung wurden zwei Aufbereitungsmethoden des Bullenspermas - Percoll-Gradient und Swim-up durch Hyaluronsäure - miteinander verglichen. Dabei wurden die Qualität des Spermas und seine Befruchtungsfähigkeit bei der In-vitro-Fertilisation (IVF) bewertet. Die tiefgefrorenen und dann aufgetauten Samenproben von 5 Simmentaler-Bullen wurden vereint und nach der Durchführung beider Aufbereitungsmethoden wurden folgende spermatologische Parameter bestimmt: Spermienmotilität (subjektiv), Spermienkonzentration (Zählkammer nach Thoma), Plasmamembranintegrität (HOS- und SYBR-14/PI-Test) und akrosomaler Status (EthD-1/FITC-PSA-Färbetest). Das Sperma wurde für die IVF mittels zweier Methoden aufbereitet: Percoll-Gradient-Zentrifugation und Swim-up-Verfahren durch Hyaluronsäure. Für die In-vitro-Fertilisation wurden pro Methode durchschnittlich 41 gereifte Oozyten verwendet, die mit den mit beiden Methoden aufbereiteten Spermien befruchtet wurden. Der Versuch wurde sechsmal wiederholt. Die Embryos wurden in einem definierten SOF/BSA-Medium bis zum 10. Tag gezüchtet. Die statistische Auswertung der Untersuchungsergebnisse erfolgte mittels ANOVA-Methode wobei die angulare Transformation (arcsin√ P/100) von in Prozenten ausgedrückten Untersuchungswerten verwendet wurde. Beim Vergleich beider untersuchten Spermienaufbereitungsmethoden in Bezug auf ihre Effizienz für die In-vitro-Fertilisation wurde festgestellt, dass keine signifikanten Unterschiede (P >0, 05) zwischen beiden Methoden bestehen, was bedeutet, dass das mit jeder dieser Methoden aufbereitete Sperma für In-vitro-Fertilisation gleich geeignet ist. Wir konnten ebenfalls keine hochsignifikante Korrelation zwischen spermatologischen Parametern und dem IVF-Erfolg feststellen (r< 0, 5 ; P >0, 05).

Metode asistirane reprodukcije kao što su umjetno osjemenjivanje, embriotransfer te oplodnja in vitro sustavno se primjenjuju u uzgojnim programima diljem svijeta omogućavajući brzi genetski napredak stada. Ove metode detaljno su proučene na domaćim životinjama, no sve se češće učinkovito primjenjuju i za očuvanje genoma ugroženih divljih vrsta i autohtonih pasmina. Na hrvatskom prostoru ugrožene autohtone pasmine preživača su: istarsko govedo (boškarin), slavonsko-srijemski podolac, buša te neke naše autohtone pasmine ovaca: cigaja, dubrovačka ruda i istarska pramenka. Primjena ovih metoda, u kombinaciji s očuvanjem stada in situ, nudi rješenje za očuvanje genetske raznolikosti naših jedinstvenih autohtonih pasmina goveda i ovaca te kontrolirani uzgoj i povećanje populacije jelenske divljači.

Proizvodnja zametaka in vitro danas se u veterinarskoj medicini primjenjuje u mnogim zemljama s rezultatima koji obećavaju. Prepoznavajući brojne prednosti ove napredne biotehnološke metode, a u cilju što brže selekcije genetski vrijednih jedinki u hrvatskom govedarstvu, Laboratorij za asistiranu reproduckiju Veterinarskog fakulteta Sveučilišta u Zagrebu počeo je još 1999. godine s primjenom proizvodnje goveđih zametaka in vitro. Temeljna istraživanja provedena u našem Laboratoriju rezultirala su proizvodnjom zametaka zadovoljavajuće kvalitete i rođenjem prvog teleta nakon transfera jajne stanice krave oplođene i uzgojene in vitro. To omogućuje širu praktičnu primjenu ove biotehnološke metode u hrvatskom stočarstvu.

Istraživanje smo proveli radi usporedbe dvije različite metode pripreme sperme za oplodnju in vitro (IVF). Ocjenjivali smo kvalitetu sperme i njihov učinak na IVF. Smrznuta/odmrznuta sperma 5 bikova Simentalske pasmine se pomiješala. Neposredno nakon odmrzavanja te nakon metoda pripreme sperme ocjenjivani su slijedeći parametri: pokretljivost (subjektivno), koncentracija (Thomina komorica), integritet membrane (HOS) i status akrosome (EthD-1/FITC). Sperma je pripremana za IVF dvjema različitim metodama: centrifugiranjem na gradijentu Percoll-a (45%-90%) i swim-up postupkom. Po metodi se prosječno koristila 41 in vitro dozrela jajna stanica, a pokus je ponovljen 6 puta. Jajne stanice su oplođene sa smrznutom/odmrznutom spermom pripremljenom dvjema metodama. Zametci su uzgajani u definiranom SOFaaBSA hranilištu do 10-og dana. Rezultati istraživanja obrađeni su statistički metodom ANOVA koristeći angularnu transformaciju (arcsin√ P/100) vrijednosti rezultata izraženih u postocima. Ustanovili smo signifikantne razlike (p0, 05) između metoda ocjenjujući rezultate IVF-a, što znači da je sperma pripremljena ovim metodama jednako kvalitetna za IVF. Ustanovili smo jake, pozitivne i signifikantne korelacije između parametara sperme (r>0, 5 ; p0, 05) te između rezultata IVF-a (r>0, 5 ; p0, 05) između parametara sperme i rezultata IVF-a.

Simmental purebreed cattle is grown for meat and milk purpose in the farms of Croatia. Eighty percent of the herd is under selection programes and subjected to AI. The recent adoption of in vitro technologies with the aim to improve our selection possibilities put a new chalenge: test our sires for the in vitro fertilization. In this phase of the program we screened nine sires of proved field fertility where we studied the thawed sperm and the selected population of spermatozoides for in vitro fertilization and the results of the IVF. The sperm is routinelly frozen in a TRIS-Citrate-Egg yolk-Glycerol diluent, in 0.5 ml straws. The non return rates of those bulls ranges form 58 to 68 %. Four straws of the same day of production were thawed, at 39°C for 1 minute, for each of three repetitions per bull, each day were tested 4 bulls. Concentration was determined in a Thoma chamber. Motility was evaluated subjectively under phase-contrast microscop. The sperm membrane integrity were evaluated using the HOS Test. The status of the acrosome were evaluated using the Trypan blue-Giemsa stain under magnification x 400. Evaluations were made before and after sperm separation. The sperm separation and clean up for IVF were performed trough a discontinuous Percoll gradient centrifugation (45-90 % Percoll in TALP withouth Ca). The IVF was made in medium with 10 mg/mL Heparine. The final concentration of the sperm suspension was adjusted to 1x106 spermatozoa/ml in the mIVF with oocytes. Oocytes were recovered from abatoir derived Simmental ovaries. A total number of 1244 oocytes were fertilized. Cleaved embryos were cultured in vitro in SOFaaBSA medium untill the Day 9. On the Day 2 we registered the number of cleaved embryos, on Day 7 the total number of morulas (M) and blastocysts (Bl) and finally the number of hatched blastocysts (hBl) on Day 9. The statistical comparison between sires was done with ANOVA using the arcsin transformation of the percent values. The average parameters before and after Percoll separation show some differences: concentration of 67 vs 51x 106 (SD: 21 vs 18) spermatozoa/ml (P

Simmental purebreed cattle is grown for meat and milk purpose in the farms of Croatia. Eighty percent of the herd is under selection programes and subjected to A.I.. The recent adoption of in vitro technologies with aim to improve our selection possibilities put a new chalenge: test our sires for the in vitro fertilization. In the first phase of this program we tested three protocols for sperm separation and clean-up for in vitro fertilization. The sperm is routinelly frozen in the Center for Reproduction and Animals Breeding of Croatia in a TRIS-Citrate-Egg yolk-Glycerol diluent, in 0.5 ml straws. Only straws with more than 50% post-thaw motility were stored for A.I. For this evaluation one sire was selected. The non return rate of this bull is 66.6 % over 2, 910 inseminations. Four straws were thawed for each of five repetitions and the motility, concentration, membrane integrity and acrosome status were evaluated before and after sperm separation. Straws were thawed at 39°C for 1 minute, and the concentration was determined in a Thoma chamber. Motility was evaluated subjectively by a trained evaluator under phase-contrast microscop. The sperm membrane integrity were evaluated using the HOS Test. The status of the acrosome were evaluated using the Trypan blue-Giemsa stain and the total number of spermatozoa with intact acrosome were counted under 400X magnification . The sperm separation and clean up for IVF were performed with three different protocols. a) Dilution and Washing of the thawed sperm in Tyrode medium supplemented with Pyruvate and BSA and without Calcium (Medium without Calcium: M-Ca), two washings with 10 and 5 ml of medium respectively, centrifugated at 200 x g for 5 min, the pellet was resuspended in medium for fertilization (mIVF: Tyrode supplemented with BSA, Penicillamine, Epinefrine, Heparine, Pyruvate and Caffeine). b) Swim up protocol: in M-Ca (1 ml, one hour at 39°C), the upper fraction of 1 ml was collected and washed twice with 3 ml of M-Ca and resuspended with mIVF. c) Discontinuous Percoll gradient centrifugation (45-90 % Percoll in M-Ca), centrifugated at 700 x g for 15 min, washed in M-Ca, and resuspended in mIVF. The final concentration of the sperm suspension was adjusted to 1x106 spermatozoa/ml in the mIVF with oocytes. An average of 40 in vitro matured oocytes were used for each of five repetitions. The oocytes were fertilized with the separated frozen/thawed bull semen. Cleaved embryos were cultured in vitro in SOFaaBSA medium till the Day 10. On the 2nd day after IVF we registered the number of cleaved embryos, the total number of morulas (M) and blastocysts (Bl) on day 7 and finally the number of hatched blastocysts (hBl) on day 10. The statistical comparison between protocols was done with ANOVA (GraphPad, Instat, V2.00) using the arcsin transformation of the percent values. The initial parameters of the thawed sperm were: 105 x 106 (&plusmn ; ; ; 27) spermatozoa/ml and 70 % (&plusmn ; ; ; 7.1) progressive motility, 51 % (&plusmn ; ; ; 19.8) membrane integrity and 12.5 % (&plusmn ; ; ; 2.12) of spermatozoa with intact acrosome. Table 1. Comparison of three sperm separation protocols: final sperm quantity and quality and IVP results. SeparationProtocol Motility%(&plusmn ; ; ; S.D.) Concentration x106 sp./ml (&plusmn ; ; ; S.D.) Membrane Integrity% (&plusmn ; ; ; S.D.) IntactAcrosome% (&plusmn ; ; ; S.D.) CleavageDay 2% (&plusmn ; ; ; S.D.) M + Bl Day 7% (&plusmn ; ; ; S.D.) hBl Day 10% (&plusmn ; ; ; S.D.) Washing 62 (7.6) 35.4 (10.4)a 60.5 (0.7) 48 (2.8) 76.6 (12.2) 22.4 (8.2) 11.8 (6.5) Swim up 65 (10.0) 11.0 (2.7)b 55 (4.2) 14 (5.7) 73.6 (16.6) 24.8 (17.9) 11.7 (10.4) Percoll 69 (7.4) 70.0 (16.5)c 55.5 (12) 22.5 (14.9) 67.5 (13.8) 20.9 (8.8) 10.8 (7.6) Values in the same column with different superscripts differ significantly (P

The present study was conducted to assess the embryo developmental competence of oocytes recovered from slaugtherhouse ovaries (SO) and those aspirated from cycling cows using transvaginal ultrasound ovum pick-up technique (OPU). The study was conducted during September 2000 year. Cumulus oocyte complexes (COCs) were collected from ovaries of slaughtered Simmental cows. Aspiration of antral follicles was performed within 2 to 4 hours after slaughter. The transvaginal ultrasound-guided (Pie Medical, the Netherlands) oocyte collection was performed in four cycling Simental donor cows, twice a week for four consecutive weeks (8 OPU sessions per cow). Oocytes were matured in vitro in TCM 199 bicarbonate medium supplemented with 10% FSC, FSH/LH (PergonalŇ 75/75 I.U., Serono), 1 mg/ml estradiol-17b and 100 mM cisteamine. After 24 hours of incubation at 39oC and with 5% CO2 in air, matured oocytes were fertilized in vitro with frozen/thawed bull semen prepared on discontinous Percoll gradient. Cleaved embryos were cultured in vitro in SOFaaBSA medium at 39oC in atmosphere of 5% CO2, 7% O2 and 88% N2 till the Day 10. The medium was changed every second day. Cultured embryos were evaluated morphologically according to IETS standards. Results are presented as mean percentage value (&plusmn ; S.E.M.) and Chi-Square Test was used to compare the developmental competence of SO and OPU-derived embryos. A total of 198 Grade 1 and 2 oocytes were selected for IVM from 30 SO ovaries. In 8 OPU sessions from four cycling donor cows a total of 89 G1 and G2 oocytes were aspirated. Fertilization rate was 79.6 &plusmn ; 6.7% for SO and 71.7 &plusmn ; 4.2% for OPU-derived oocytes. Embryos cultured in vitro from OPU-derived oocytes developed to the morula/blastocyst stage (Day 7) at significantly higher rate than SO-derived oocytes (51.0 &plusmn ; 4.3% vs 33.4 &plusmn ; 5.9%, P

Na ekonomiji u Križevačkom Lemešu rođeno je prvo vitalno žensko tele nakon transfera jajne stanice krave oplođene in vitro, u četvrtak 3. svibnja 2001. Jajne stanice krave davateljice dobivene su aspiracijom antralnih folikula iz jajnika sakupljenih na liniji klanja te stavljene u postupak dozrijevanja in vitro. Nakon 24 sata dozrijevanja u 5% CO2 te na 39oC jajne stanice su oplođene in vitro duboko zamrznutom/odmrznutom spermom bika, pripremljenom na gradijentu Percolla. Konačna koncentracija spermija podešena je na 106 spermija po mililitru. Oplođene jajne stanice uzgajane su in vitro do stadija blastociste u definiranom mediju koji je mijenjan svaki drugi dan, u atmosferi s 5% CO2, 7% O2, i 88% N2. Najkvalitetniji zameci transferirani su u dvije krave primateljice simentalnske pasmine, sinkronizirane dvokratnom aplikacijom PGF2α , u kojih je 7. dan nakon ovulacije na jajnicima utvrđeno kvalitetno žuto tijelo. Prijenos zametaka u maternicu primateljice izveden je pod epiduralnom anestezijom, a zameci su transferirani u prednju trećinu roga na kojoj je bilo žuto tijelo. Kod jedne krave primateljice gravidnost je dijagnosticirana ultrazvukom 25. dana nakon transfera, te potvrđen nakon 2 mjeseca. Rođenjem prvog "teleta iz epruvete"zaokružena su višegodišnja istraživanja na projektima Ministarstva znanosti i tehnologije "Embriotransfer u krava" te "In vitro oplodnja u goveda" glavnog istraživača prof. dr.sc. Zdenka Makeka. U okviru tih projekata utemjeljen je Laboratorij za asistiranu reprodukciju pri Klinici za porodništvo i reprodukciju Veterinarskog Fakulteta Sveučilišta u Zagrebu gdje je proveden cijeli postupak dozrijevanja, oplodnje i uzgoja goveđih zametaka in vitro. U radu laboratorija, kojim upravlja voditeljica mr.sc. Iva Getz, dr.vet.med., sudjeluju svi djelatnici Klinike te dr.sc. Mario Matković, suradnik na projektu iz Centra za reprodukciju u stočarstvu Hrvatske (CRSH). Transfer zametaka u krave primateljice obavio je Zvonko Tuček, dr.vet.med., djelatnik CRSH.

Tijekom istraživanja na projektima Ministarstva znanosti i tehnologije "Embriotransfer u krava" i "In vitro oplodnja u goveda" glavnog istraživača prof. dr. sc. Zdenka Makeka, 8. srpnja 1992. godine pri Klinici za porodništvo i reprodukciju Veterinarskog Fakulteta Sveučilišta u Zagrebu utemeljen je Laboratorij za asistiranu reprodukciju. U Laboratoriju je postavljen standardni postupak oplodnje i uzgoja goveđih zametaka in vitro te su time stvoreni preduvjeti za primjenu i korištenje prednosti ovih metoda u našoj stočarskoj proizvodnji. In vitro proizvodnja u govedarstvu ima brojne primjene kao što su zaobilaženje nepredvidivog superovulacijskog odgovora i nekih poremećaja u spolnim organima davateljica, proizvodnja zametaka iz jajnih stanica dobivenih ultrazvučnom punkcijom jajnika, kontrola i sprečavanje širenja zaraznih bolesti, skraćenje generacijskog intervala proizvodnjom zametaka od ženske teladi prije puberteta, dobivanje tovne teladi transferom zametaka u mliječne krave, dijeljenje zametaka u svrhu dobivanja blizanaca, utvrđivanje plodnosti bikova koji se koriste za U.O. i ubrzanje postupka progenog testiranja bikova te drugo. Primjena navedenih biotehnoloških metoda nužna je za uključivanje naše stočarske proizvodnje u međunarodne tijekove, pogotovo ulaskom Republike Hrvatske u WTO. Europska stočarska proizvodnja je ugrožena zbog širenja stočnih zaraznih bolesti kao što su slinavka i šap te goveđa spongiformna encefalopatija. Republika Hrvatska ima kvalitetan stočni fond i još uvijek uživa status zemlje slobodne od danas najopasnijih stočnih zaraza, a kako su rijetke zemlje koje imaju mogućnosti proizvodnje i izvoza zdrave hrane to je prednost koju svakako moramo iskoristiti. Formiranjem matičnih stada domaćeg simentalskog goveda te korištenjem poželjnog genoma za proizvodnju mesa i mlijeka izravno se potiče širenje boljih proizvodnih svojstava životinja naše populacije čime ostaje očuvan status zdravog proizvoda. Važno je naglasiti da su životinje u postupcima asistirane reprodukcije pod strogom veterinarskom i sanitarnom kontrolom čime je mogućnost prijenosa bolesti svedena na najmanju moguću mjeru. Prema tome, ne samo da imamo mogućnost odabira i širenja genoma najkvalitetnijih životinja, nego imamo mogućnost kontrole zdravlja životinja i ljudi te dobivanja konačnog proizvoda slobodnog od aktualnih stočnih zaraza.

Biotehnologija rasplođivanja podrazumijeva zahvate kojima u kontroliranim uvjetima rasplođujemo životinje i utječemo na genetsku selekciju širenjem poželjne genetike prema željenim proizvodnim svojstvima. Te biotehnologije razvijaju se posljednjih 50 godina prema potrebama i mogućnostima svakog razdoblja. Prva generacija biotehnologije bilo je umjetno osjemenjivanje, druga embriotransfer i srodne tehnologije, treća proizvodnja zametaka in vitro, a četvrta manipulacija genomom uz pomoć transgeneze i kloniranja. Primjena biotehnologije u rasplođivanju životinja omogućuje 1. genetski napredak stada dobivanjem velikog broja potomaka od najkvalitetnih životinja, 2. međunardoni transport i trgovinu zamrznutim zamecima te tako smanjuje rizik prenošenja zaraznih bolesti jer više nije potreban transport živih životinja, 3. povećava ponudu visokokvalitetne stoke, 4. omogućuje aplikaciju drugih tehnologija koje imaju dodatne primjene, kao npr. seksiranje i bližnjenje zametaka, genetske pretrage, seksiranje sperme, proizvodnja transgene stoke i drugo. Ti biotehnološki zahvati zahtijevaju sofisticiranu opremu i specijalnu obuku te su stoga skupi za široku primjenu, ali je njihov učinak na odabranoj stoci velik i brz. Uglavnom svi koji rade u tom području slažu se da je vrlo važno što brže prenijeti genom od matičnih stada u proizvodne jedinice. U sklopu projekta Ministarstva znanosti i tehnologije RH "Proizvodnja in vitro goveđih zametaka" postavljen je standardni protokol za dozrijevanje, oplodnju i uzgoj zametaka in vitro. Također je osnovan Laboratorij za asistiranu reprodukciju životinja pri Klinici za porodništvo i reprodukciju Veterinarskog fakulteta Sveučilišta u Zagrebu, a u suradnji sa stručnjacima Centra za reprodukciju u stočarstvu Hrvatske d.o.o. Zameci proizvedeni u našem laboratoriju zadovoljavaju kriterije za embriotransfer. U radu je iznesen kratki prikaz prednosti primjena biotehnoloških metoda u uzgoju životinja te su predložene moguće primjene u našim uvjetima.

Biotehnološke metode, embriotransfer i manipulacija zametcima in vitro, primjenjuju se u razvijenim zemaljama kao sastavni dio rasplođivanja životinja. Intenzivnim istraživanjima značajno su poboljšani rezultati in vitro dozrijevanja, oplodnje i uzgoja goveđih zametaka. Da bi se ova tehnologija mogla u potpunosti iskoristiti predložene su metode aspiracije jajnih stanica za IVF iz odabranih živih davateljica. Opisana je metoda transvaginalne ultrazvučne punkcije jajnika (OPU = transvaginal ovum pick-up) u dvije krave davateljice nakon prethodne stimulacije jajnika s FSH/LH (Stimufol, Merial, France) tijekom tri uzastopna dana. Punkcija folikula obavljena je četvrtog dana, a postupak je ponovljen tijekom tri uzastopna tjedna. OPU sistem (Pie Medical, Nizozemska) sastoji se od tri komponente: ultrazvučnog aparata sa sektorskom sondom od 5/7,5 Mhz, aspiracijske pumpe i sustava za uvođenje igle. Nakon sedacije i epiduralne anestezije davateljica obavljena je aspiracija folikula pod kontrolom ultrazvuka. Broj nezrelih jajnih stanica (kumulus-oocita kompleks = KOK) dobivenih ultrazvučnom punkcijom jajnika kretao se od 1 do 6 (prosječno 3,5); uspjeh aspiracije iznosio je u prosjeku 40,7%. Prosječan postotak jajnih stanica oplođenih in vitro, nakon 24 sata koinkubacije sa spermijima, iznosio je 54%. Sedmog dana uzgoja in vitro u definiranom mediju dobili smo prosječno 33% morula i blastocista dobre kvalitete, pogodne za transfer ili duboko zamrzavanje.

U radu su opisane i uspoređene dvije metode uzgoja in vitro goveđih zametaka: uzgoj u kokulturi sa stanicama granuloze i u SOFaaBSA definiranom mediju. Aspiracijom antralnih folikula iz 60 jajnika junica i krava iz klaonice dobili smo 428 nezrelih jajnih stanica za dozrijevanje in vitro. Nakon 24 sata inkubacije postotak dozrijevanja iznosio je 98%. Jajne stanice oplođene su in vitro duboko zamrznutom/odmrznutom spermom bika, pripremljenom na gradijentu Percolla. Postotak brazdanja nakon oplodnje iznosio je 63% i 70% (u kokulturi, odnosno SOF-u). Nakon 7 dana uzgoja in vitro dobiveno je 27% i 29% morula i blastocista, a 11. dana 12% i 24% te 1% i 12% izljuštenih blastocista (u kokulturi, odnosno SOF-u). Dobiveni zameci su dobre kvalitete te zadovoljavaju uvjete za embriotransfer. Zametci dobiveni uzgojem u SOFaaBSA definiranom mediju pokazivali su statistički signifikantno bolje rezultate 11. dana uzgoja. Na temelju dobivenih rezultata odlučili smo zamijeniti naš standardni protokol uzgoja u kokulturi sa stanicama granuloze sa uzgojem in vitro u definiranom sistemu SOFaaBSA.

The use of biotechnology, and therefore embryo transfer and manipulation with embryos in vitro, has became an integral part of animal reproduction. In order to further exploit this technique several oocyte aspiration methods for IVF from previously selected living cows are suggested. A method is described for transvaginal ovum pick-up (OPU) in two donor cows, stimulated with FHS/LH (Stimufol , Merial, France) for 3 consecutive days. Aspiration of the follicles was performed on Day 4 and procedure was repeated once a week for three consecutive weeks. The OPU system (Pie Medical, the Netherlands) consists of 3 components: an ultrasound scanner with a sector 5/7, 5 MHz tranducer, an aspiration pump and a needle guidance system. Donor cows were sedated and after additional relaxation by epidural anesthesia, follicles were punctured under echographic control. The number of oocytes (cumulus-oocyte complex = COC) collected using OPU varied from 1 to 6 (2.5 an average) and mean recovery rate was 44.1%. Fertilization rate after 24 hours of oocytes-sperm co-incubation was 53.3%. Culture in vitro in SOFaaBSA media rendered an average of 33.3% morulas and blastocysts of good quality, for ET or cryopreservation.

Since the first calf as a result of in vitro fertilization was born in 1982, the in vitro technology has advanced enormously. In vitro production (IVP) of bovine embryos has become a routine procedure in many laboratories. It is now in the process of transition from strictly scientific studies to a technology which permits breeding plans and implementation of other embryotechnologies such as freezing, twining, cloning, transgenesis, sexing of embryos and others. Therefore, three years ago we started a project "In vitro fertilization in cows", with the aim to establish the basic protocol for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of bovine oocytes and to introduce this new biotechnology in our veterinary medicine and breeding programs. Cumulus oocyte complexes (COCs) were collected from ovaries of slaughtered cows by aspiration of antral follicles with diameter less than 8 mm. A total of 426 oocytes were selected for IVM from 158 ovaries (2.7 oocytes per ovary, i.e. 5.4 per pair). The oocytes were matured in drops of maturation medium (TCM 199 medium supplemented with fetal calf serum and hormones). After 24 hours of incubation at 39oC and with 5% CO2, matured oocytes were inseminated in vitro with frozen/thawed bull semen, prepared by gradient separation. Forty-eight hours after insemination 4- and 8-cell embryos were selected and cultured in vitro to the blastocyst stage in supplemented TCM 199 medium in co-culture with granulosa cells. Maturation rate was determined based on expansion of cumulus and corona cells and it was 87.6%. Fertilization i.e. cleavage rate determined 48 hours after insemination was 56.3% and blastocysts production rate observed on day 7 was 14.7%. Bovine blastocysts produced in vitro were of good quality, according to IETS standards and suitable for embryo transfer or cryopreservation. The next programmed step is the ultrasound-guided transvaginal ovum pick-up (OPU) from the ovaries of living cows for embryo transfer purposes.