Veise sigimine: kõrgkooliõpik

Eestis on veisekasvatusest lugu peetud juba kiviajast peale. Meil on heade tõuomadustega ja suure toodanguga piimakari ja kiiresti kasvav lihakari ning me suudame toota kvaliteetset piima ja veiseliha nii kohalikele tarbijatele kui ka ekspordiks. Veiste sigimisprobleemid on oluliseks veisekasvatuse kasumlikkust mõjutavaks teguriks. Selleks, et sigimisprobleemide olemust mõista ja nende teket ennetada või neid ravida, on vajalikud põhjalikud teadmised veise sigimise erinevatest aspektidest. Seni puudub üks terviklik veise sigimise alast teavet koondav eestikeelne õppevahend. Käesoleva õpiku abil soovime seda lünka täita. Oleme õpikusse kokku kogunud teadmised suguorganite morfoloogiast ja füsioloogiast, tiinusest ja sünnitusest ning poegimisjärgsetest haigustest. Suguorganite anatoomiat käsitletakse õpikus mitte klassikalise, vaid funktsionaalse anatoomia seisukohast. Veise tiinuse ja sünnituse

Genome-wide histone modification profiling of inner cell mass and trophectoderm of bovine blastocysts by RAT-ChIP

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin-related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited. Here we describe a novel method called Restriction Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables global histone modification profiling from as few as 100 cells. The method is simple, cost-effective and takes a single day to complete. We demonstrate the sensitivity of the method by deriving the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass and trophectoderm of bovine blastocyst stage embryos.Peer reviewe

Veise sigimine : kõrgkooliõpik

KõrgkooliõpikÜlle Jaakma ja Mihkel Jalakase toimetatud õpik „Veise sigimine“ valiti 2018. aasta parimaks eestikeelseks kõrgkooliõpikuks.Eestis on veisekasvatusest lugu peetud juba kiviajast peale. Meil on heade tõuomadustega ja suure toodanguga piimakari ja kiiresti kasvav lihakari ning me suudame toota kvaliteetset piima ja veiseliha nii kohalikele tarbijatele kui ka ekspordiks. Veiste sigimisprobleemid on oluliseks veisekasvatuse kasumlikkust mõjutavaks teguriks. Selleks, et sigimisprobleemide olemust mõista ja nende teket ennetada või neid ravida, on vajalikud põhjalikud teadmised veise sigimise erinevatest aspektidest. Seni puudub üks terviklik veise sigimise alast teavet koondav eestikeelne õppevahend. Käesoleva õpiku abil soovime seda lünka täita. Oleme õpikusse kokku kogunud teadmised suguorganite morfoloogiast ja füsioloogiast, tiinusest ja sünnitusest ning poegimisjärgsetest haigustest. Suguorganite anatoomiat käsitletakse õpikus mitte klassikalise, vaid funktsionaalse anatoomia seisukohast. Veise tiinuse ja sünnituse patoloogia osas antakse ülevaade ka väärarendite ja abordi problemaatikast. Esmakordselt käsitletakse eestikeelses õpikus kaasaegset sigimise biotehnoloogiat, sh embrüotehnoloogiat ja transgeenset tehnoloogiat, koos võimalike rakendustega aretustöös. Õpik on mõeldud eeskätt veterinaarmeditsiini eriala üliõpilastele, aga seda saavad käsiraamatuna kasutada ka praktiseerivad loomaarstid, seemendustehnikud ja loomakasvatuse spetsialistid. Lisaks sellele saavad siit informatsiooni bioloogia eriala üliõpilased ja biotehnoloogia ning kliinilise diagnostika spetsialistid, samuti bioloogiaõpetajad. Õpiku autorid on õppejõud ja teadlased, kes on oma kitsama valdkonna tunnustatud asjatundjad ning puutuvad oma töös käsitletud teemade ringi puudutavate probleemidega kokku iga päev. Täname kõiki autoreid nende suure panuse eest õpiku valmimisse. Suur tänu Eha Järvele, kes on rahulikult ja asjatundlikult aidanud eri autorite kirjutatud osad üheks tervikuks ühendada. Oleme tänulikud kõigile kolleegidele, kes oma tähelepanekute ja soovitustega on selle raamatu valmimisel kaasa aidanud. Loodame, et õpik pakub nii õppuritele kui ka praktikutele kasulikku ja vajalikku informatsiooni. Ülle Jaakma õpiku toimetaj

Assessing the effects of bovine embryo-derived extracellular vesicles on the development of individually cultured bovine embryos

In vitro embryo production requires an enriched microenvironment with various vital cell-secreted factors. In vitro cultured single bovine embryos have demonstrated lower blastocyst rate compared to grouped cultured embryos. We assumed that extracellular vesicles (EVs) within an embryo culture system may affect normal in vitro development. This study aimed to assess the supplementation effects of bovine embryo-derived EVs on the development of individually cultured bovine embryos. Bovine oocytes were in vitro maturated (IVM) for 24 h and then in vitro fertilized (IVF). In preliminary experiments, we established that group cultured embryos in EV depleted Bovine Serum Albumin (BSA) media successfully completed their development; while single cultured embryos were only able to reach the morula stage and then degenerated. Hence, we tested EVs supplementation effects in droplets of EV depleted BSA media covered by mineral oil. EVs used for supplementation were produced from single embryos cultured for 8 days in droplets of BSA culture media under mineral oil. Conditioned medium was collected on day 5. EVs were purified, using Izon columns, from embryos which reached the blastocyst stage and embryos which cleaved on day 2 then degenerated. Non-EV supplemented single embryos cultured in BSA media were considered as control. Purified EVs were characterized by nanoparticle tracking analysis and transmission electron microscope (TEM). A total of 8.8 ×106 particles/ml, which we assumed to be the approximate amount of EVs that a single embryo may release during in vitro culture, was supplemented to each droplet on day 4 post-fertilization. Cleavage rates were 70 and 80% for the supplemented groups and 86% for the control. Morula rates were 40%, 47%, and 47% respectively. No blastocyst was observed within the supplemented groups while the control group counted 33% of blastocysts. Our study suggests that BSA EVs support single cultured embryos to complete their development and that a single embryo needs a significant amount of EVs to reach the blastocyst stage. More researches are needed to understand the role of culture media EVs in supporting single embryo development

A dual colour FISH method for routine validation of sexed Bos taurus semen

Background: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.Peer reviewe

A dual colour FISH method for routine validation of sexed Bos taurus semen

Abstract Background Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p Peer reviewe

Specific trophoblast transcripts transferred by extracellular vesicles affect gene expression in endometrial epithelial cells and may have a role in embryo-maternal crosstalk

Background Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA. Methods We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR. Results We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication. Conclusion Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.Peer reviewe

Production of calves following nonsurgical transfer of fresh and refrigerated bovine demi-embryos

Alkionhalkaisun ja -siirron tehokkuutta selvitettiin kahdessa kokeessa siirtämällä halkaistuja alkioita tuoreina tai yön yli säilytyksen (4°C) jälkeen. Alkionhalkaisut suoritettiin yksinkertaisella mikroveitsitekniikalla maatilalla. Kokeessa 1 siirrettiin 28 alkion puolikasta tuoreena vastaanottajahiehoihin, joista 20 tiinehtyi (71,4 %) tuottaen 19 vasikkaa. Kylmäsäilytyksen jälkeen (kokonaissäilytysaika 17-18 tuntia) 14 alkion puolikkaasta 12 arvioitiin siirtokelpoisiksi. Näistä yhdeksän siirrettiin vastaanottajahiehoihin, joista viisi tiinehtyi tuottaen neljä vasikkaa. Kokeessa 2 siirrettiin 21 alkion puolikasta vastaanottajahiehoihin. Näistä 12 tiinehtyi (57,1 %) poikien 10 vasikkaa. Kylmäsäilytyksen (kokonaissäilytysaika 22-24 tuntia) jälkeen siirrettiin 19 alkion puolikasta hiehoihin, joista viisi tiinehtyi. Näistä neljä poiki vasikan. Tulosten perusteella voidaan todeta, että yksinkertaisella tekniikalla on mahdollista tuottaa halkaistuja alkioita, joiden elinkyky alkionsiirron jälkeen on samaa luokkaa kuin mitä yleensä saadaan käsittelemättömillä alkioilla. Kylmäsäilytyksen jälkeen halkaistujen alkioiden elinkyky heikkenee selvästi, mutta halkaistujen alkioiden kylmäsäilytys voi joissakin tilanteissa olla käytännöllistä. Toisen puolikkaan voi siirtää vastaanottajalle heti halkaisun jälkeen ja toisen seuraavana päivänä kylmäkuljetuksen jälkeen esimerkiksi toisella paikkakunnalla. Toinen menetelmän sovellutus on ottaa alkioista koepaloja, lähettää ne laboratorioon analysoitavaksi, ja valita diagnoosituloksen perusteella, mitkä kylmäsäilytetyistä alkioista siirretään seuraavana päivänä.The viability of microblade-bisected Day-7 embryos transferred to recipients without zona pellucidae directly after splitting or after overnight storage at 4°C was investigated in two experiments. In Experiment 1, 26 demi-embryos of excellent or good quality and two of fair quality were transferred to 28 heifer recipients 1-3 h after splitting. The transfers resulted in 20 pregnancies (71.4% pregnancy rate), and 19 calves were born (one pregnant heifer was slaughtered). Another 14 demi-embryos were cultured 1 to 3 h at room temperature, transported for 3 h at 8-10°C and stored for 12 h in a refigerator at 4°C. Twelve of the 14 demi-embryos were considered transferable after storage and nine of these were transferred to nine recipients, of which five became pregnant. Four live calves were born. In Experiment 2, 21 excellent or good quality demi-embryos were transferred into 21 heifer recipients to produce 12 pregnancies (57.1%) and 10 live calves. Another 19 demi-embryos (17 excellent to good quality and two fair quality) were transferred after storage for 1-2 h at 8°C and 18 h at 4°C. Five recipients became pregnant, of which four delivered live calves. It is concluded that a high pregnancy rate can be achieved after the transfer of fresh demi-embryos produced by a simple method of embryo bisection under farm conditions. Although the transfer of demi-embryos stored overnight at 4°C results in decreased pregnancy rates, refrigeration of demi-embryos may be useful in certain practical situations. However, further experiments are needed to determine the optimal conditions for overnight storage of demi-embryos.vokKirjasto Aj-kVasikoiden tuottaminen tuoreilla ja kylmäsäilytetyillä halkaistuilla alkioill

Vasikoiden tuottaminen tuoreilla ja kylmäsäilytetyillä halkaistuilla alkioilla

The viability of microblade-bisected Day-7 embryos transferred to recipients without zona pellucidae directly after splitting or after overnight storage at 4°C was investigated in two experiments. In Experiment 1, 26 demi-embryos of excellent or good quality and two of fair quality were transferred to 28 heifer recipients 1-3 h after splitting. The transfers resulted in 20 pregnancies (71.4% pregnancy rate), and 19 calves were born (one pregnant heifer was slaughtered). Another 14 demi-embryos were cultured 1 to 3 h at room temperature, transported for 3 h at 8-10°C and stored for 12 h in a refigerator at 4°C, Twelve of the 14 demi-embryos were considered transferable after storage and nine of these were transferred to nine recipients, of which five became pregnant. Four live calves were born. In Experiment 2, 21 excellent or good quality demi-embryos were transferred into 21 heifer recipients to produce 12 pregnancies (57.1%) and 10 live calves. Another 19 demi-embryos (17 excellent to good quality and two fair quality) were transferred after storage for 1-2 h at 8°C and 18 h at 4°C. Five recipients became pregnant, of which four delivered live calves. It is concluded that a high pregnancy rate can be achieved after the transfer of fresh demi-embryos produced by a simple method of embryo bisection under farm conditions. Although the transfer of demi-embryos stored overnight at 4°C results in decreased pregnancy rates, refrigeration of demiembryos may be useful in certain practical situations. However, further experiments are needed to determine the optimal conditions for overnight storage of demi-embryos.Alkionhalkaisun ja -siirron tehokkuutta selvitettiin kahdessa kokeessa siirtämällä halkaistuja alkioita tuoreina tai yön yli säilytyksen (4°C) jälkeen. Alkionhalkaisut suoritettiin yksinkertaisella mikroveitsi-tekniikalla maatilalla. Kokeessa 1 siirrettiin 28 alkion puolikasta tuoreena vastaanottajahiehoihin, joista 20 tiinehtyi (71,4 %) tuottaen 19 vasikkaa. Kylmäsäilytyksen jälkeen (kokonaissäilytysaika 17-18 tuntia) 14 alkion puolikkaasta 12 arvioitiin siirtokelpoisiksi. Näistä yhdeksän siirrettiin vastaanottajahiehoihin, joista viisi tiinehtyi tuottaen neljä vasikkaa. Kokeessa 2 siirrettiin 21 alkion puolikasta vastaanottajahiehoihin. Näistä 12 tiinehtyi (57,1 %) poikien 10 vasikkaa, Kylmäsäilytyksen (kokonaissäilytysaika 22-24 tuntia) jälkeen siirrettiin 19 alkion puolikasta hiehoihin, joista viisi tiinehtyi. Näistä neljä poiki vasikan. Tulosten perusteella voidaan todeta, että yksinkertaisella tekniikalla on mahdollista tuottaa halkaistuja alkioita, joiden elinkyky alkionsiirron jälkeen on samaa luokkaa kuin mitä yleensä saadaan käsittelemättömillä alkioilla. Kylmäsäilytyksen jälkeen halkaistujen alkioiden elinkyky heikkenee selvästi, mutta halkaistujen alkioiden kylmäsäilytys voi joissakin tilanteissa olla käytännöllistä. Toisen puolikkaan voi siirtää vastaanottajalle heti halkaisun jälkeen ja toisen seuraavana päivänä kylmäkuljetuksen jälkeen esimerkiksi toisella paikkakunnalla. Toinen menetelmän sovellutus on ottaa alkioista koepaloja, lähettää ne laboratorioon analysoitavaksi, ja valita diagnoosituloksen perusteella, mitkä kylmäsäilytetyistä alkioista siirretään seuraavana päivänä