In Vitro Production of Embryos at Research and Development Station for Cattle Breeding Dancu, Iasi – First Bovine Embryos Produced In Vitro in North-Eastern Romania

AbstractThein vitroproduction (IVP) of bovine embryos increases the selection intensity in cattle and reduces the generation interval, which is very important in the genetic gain. In Romania, this reproductive biotechnology has shown a timid evolution in the last years, although the need for genetic improvement in the area is present. The aim of this paper is to describe the work that resulted in first bovine embryos obtained through IVP in North-Eastern Romania. Oocytes were collected by slashing ovaries from slaughtered cows, matured in a TCM199-based medium and fertilized in TL-based medium microdrops with sperm processed by swim-up procedure. The presumptive embryos were cultured one day in TCM199 and 8 days in SOF-based medium and evaluated in days 7, 8 and 9 after fertilization. We retrieved an average number of 83 usable oocytes/IVF session, which represents 73.8% from the total harvested oocytes. The average number of cleaved embryos was 50.8 per IVF, reflecting an average cleavage rate of 61.2%. An average of 8.6 blastocysts/IVF session was obtained, representing 10.4% of the selected oocytes or 16.9% of the number of cleaved embryos. Although suboptimal, the results were comparable with other reports on IVP in cattle. The adapted IVP protocol, based on maturation with TCM199, fertilization in microdrops of TL and culture of presumptive embryos one day in TCM199 and afterwards in SOF seems to offer acceptable results and will be used for further attempts to produce bovine embryos.

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162 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE IN RESISTANCE TO EMBRYO CRYOPRESERVATION

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab162 ◽

2014 ◽

Vol 26 (1) ◽

pp. 195

Author(s):

R. R. D. Maziero ◽

D. M. Paschoal ◽

M. D. Guastali ◽

M. J. Sudano ◽

P. N. Guasti ◽

...

Keyword(s):

Breeding Systems ◽

Embryo Cryopreservation ◽

Bovine Embryos ◽

In Vitro Production ◽

Cattle Breeding ◽

Meiotic Arrest ◽

Cell Groups ◽

Level Of Significance ◽

Vitro Production

In vitro embryo production by maturation, fertilization, and culture has become a valuable assisted reproductive technology in cattle breeding systems. However, even with the notable innovations in this system, the greatest obstacles for the complete success of this biotechnology are the low pregnancy rates after transfer and the greater sensitivity to cryopreservation. Thus, this study evaluated the effect of the addition of a meiotic arrest using butyrolactone I and roscovitine on in vitro production of bovine embryos. Nelore oocytes were matured in TCM-199 with Earle's salt + 10% FCS, FSH, and LH, in 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in presence of butyrolactone I (BL-I) 25 μM + roscovitine (ROS) 6.25 μM. Then, the oocytes (Control n = 405; BL-I + ROS n = 386) were cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the conditions above. Semen was selected through Percoll gradient and the concentration adjusted to 2 × 106 sperm mL–1. The presumed zygotes were culture in 90-μL droplets of SOFaa + 0.6% BSA + 2.5% FCS in 5% CO2 until Day 7, when the blastocyst rate was evaluated. We performed 5 replicates. Data were analysed with ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. Statistical differences were observed in blastocyst production rate: Control: 42.64% and BL-I + ROS: 55.96% (P < 0.05). The remaining embryos (Control n = 163; BL-I + ROS n = 182) were vitrified, warmed, and recultured in SOFaa with 10% FCS for 12 h. After this period, re-expansion rate was determined. We found difference in re-expansion rates of groups (Control: 84.66% and BL-I + ROS: 93.40%; P < 0.05). Additionally, differences were found among total number cell groups: Control: 54.33 and BL-I + ROS: 97.5; P < 0.05). The use of BL-I + ROS is able to better a producing rate of in vitro embryos and improve the cryotolerance. The authors acknowledge FAPESP 2010/18994-4 and 2011/08926-4 for financial support.

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In vitro production of bovine embryos

Veterinarski glasnik ◽

10.2298/vetgl0304257p ◽

2003 ◽

Vol 57 (3-4) ◽

pp. 257-264

Author(s):

Vojislav Pavlovic ◽

Jelena Aleksic

Keyword(s):

Pharmaceutical Products ◽

Bovine Embryos ◽

In Vitro Production ◽

Cattle Breeding ◽

Vitro Fertilization ◽

Laboratory Conditions ◽

Female Donor ◽

Vitro Production ◽

Bovine Oocytes

Biotechnology is used for the purpose of improving production, and developing animal and pharmaceutical products. In roder to achieve these objectives, it is necessary to manipulate these processes. Reproductive biotechnology can be used independently, or it can be used in connection with other techniques. Thus, for instance, successful culture of embryos in laboratory conditions is a necessary precondition for the production and creation of transgenic and cloned animals. The in vitro process of embryo production is narrowed down to three basic steps: 1. collecting oocytes form a female donor, 2. fertilization of oocytes under laboratory conditions, 3. growth of the embryo in a medium and transfer of the embryo into the recipient. The paper describes the IVP procedure (in vitro production) of bovine embryos; the advantages and shortcomings of this method, as well as possibilities for its application in cattle breeding. This technology is still quite new, so taht both the technique and the mediums are constantly being improved. The technique of fertilizing bovine oocytes, as well as their development in laboratory conditions was discovered in 1980, and the first calf produced using in vitro fertilization (IVF) was born in 1982. IVP implies a series of steps, and if just one of them is not done perfectly, the result is a small number of embryos, or even none at all.

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262 REPORTS OF IN VITRO PRODUCTION AND PREGNANCY RATES OF BOVINE EMBRYOS PRODUCED BY POST-THAWING SEXED SEMEN

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab262 ◽

2010 ◽

Vol 22 (1) ◽

pp. 288 ◽

Cited By ~ 2

Author(s):

K. B. Avelino ◽

M. R. Rossetto ◽

A. C. Maraia ◽

M. R. Lima ◽

A. T. R. Mansano ◽

...

Keyword(s):

Flow Cytometry ◽

Pregnancy Rate ◽

Pregnancy Rates ◽

Bovine Embryos ◽

In Vitro Production ◽

Cleavage Rate ◽

Genetic Value ◽

Vitro Production ◽

Fresh Semen

Births of predetermined-sex calves illustrates the importance of sperm sexing technology for in vitro production of bovine embryos (Johnson, 2000). Flow cytometry techniques for sperm sexing are usually performed on fresh semen. However, many bulls presenting high genetic value died or became unproductive before development of sperm sexing technologies. The technique of fresh semen sexing and semen reverse sorting method is based on separation of sperm according to differences in DNA content (X or Y). The objective of this study is to examine the blastocysts and pregnancy rates of bovine embryos produced by fertilization with sperm sexed with the post-thawing procedure, called reverse sorting. Oocytes were obtained by ovum pick-up from Dairy Gir, Nelore, and Guzera cows. The COC were transported to the laboratory and matured at 38.5°C and 5% of CO2 in air, TCM-199 medium with FCS (10% vol/vol), FSH (1.0 μg mL-1), hCG (50 μg mL-1), estradiol (1.0 μg mL-1), sodium pyruvate (0.20 mM), and amicacin (83.4 μg mL-1). IVF was performed 24 to 26 h after the onset of maturation. Doses of commercialized semen (3 to 5) from the specified breeds were sent to Goyaike Brazil Agropecuária Ltda. The semen sexing process was performed by flow cytometry and semen was returned to the IVF laboratory at 18°C. In the Tecgene laboratory, semen was centrifugated using Percoll gradient and evaluated. After 18 h, the presumptive zygotes were transferred to IVC in SOF medium at 38.5°C and 5% CO2 in air. The cleavage rate was evaluated 48 h after IVF and embryos were transferred at Day 7. A total of 5213 viable oocytes were obtained from 266 donors, fertilized with 7 different reverse sorted semen samples (1 Dairy Gir, 2 Nelore, and 4 Guzera) resulting in 1333 embryos. From these, 1084 transferred embryos resulted in 260 pregnancies to this time, with 226 females and 34 males. Each bull was evaluated separately according to cleavage rate, production of blastocysts, pregnancy rate, and percentage of females, respectively. Bull 1 presented 100%, 38%, 25%, and 90%; bull 2: 98%, 38%, 21%, and 90%; bull 3: 84%, 23%, 21%, and 84%; bull 4: 87%, 15%, 29%, and 86%; bull 5: 92%, 53%, 70%, and 71%; bull 6: 78%, 15%, 34%, and 71%, and bull 7: 55%, 27%, 50%, and 78%. The average of in vitro blastocysts (26%), the pregnancy rate (24%), and the percentage of females (87%) are similar to rates obtained by commercial fresh semen sexed by Goyaike Brazil Agropecuária Ltda. In conclusion, semen reverse sorting is an alternative method for selecting sex of in vitro production of bovine embryos using thawed semen collected from extinct or unproductive bulls.

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135 RECOMBINANT BOVINE SOMATOTROPIN ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab135 ◽

2014 ◽

Vol 26 (1) ◽

pp. 181

Author(s):

L. Berté ◽

L. Vasconcelos ◽

L. Hatamoto-Zervoudakis ◽

W. Yamazaki ◽

L. Yamazaki ◽

...

Keyword(s):

Bovine Embryos ◽

In Vitro Production ◽

Blastocyst Development ◽

Cleavage Rate ◽

Recombinant Bovine Somatotropin ◽

Group 4 ◽

Group 3 ◽

Group 2 ◽

Vitro Production

Bovine growth hormone (bGH) has been used to improve the results for in vitro production of bovine embryos. Inclusion of bGH in the maturation medium increases both rate of cleavage and frequency of blastocyst development. Thus, the purpose of the present study was to evaluate the effect of recombinant bovine somatotropin (rBST) on cleavage and blastocyst development of bovine embryos when included during in vitro maturation (IVM) only (Group 1), during both IVM and in vitro culture (IVC; Group 2), during IVC only (Group 3), or not included during either IVM or IVC (Group 4). Specifically, in Group 1, oocytes were matured in TCM 199 (Earle's salts) supplemented with 10% FCS, LH, FSH, oestradiol, and amikacin (IVM medium), plus 100 ng mL–1 of rBST and cultured in SOFaaci supplemented with essential amino acids, tri-sodium citrate, myo-inositol, and 5% FBS. In group 2, oocytes were matured in IVM medium containing 100 ng mL–1 of rBST and cultured in SOFaaci supplemented with essential amino acid, tri-sodium citrate, myo-inositol, 5% FBS; on Day 5, rBST (50 ng mL–1) was added. In Group 3, oocytes were matured in IVM medium without rBST; on Day 5, rBST (50 ng mL–1) was added. Group 4 was the control, without rBST supplementation. The treatment groups were analysed using the SAS® (SAS Institute Inc., Cary, NC, USA) in a completely randomised design (P < 0.05). Somatotropin has receptors in cumulus cells and in the zona pellucida acting directly in the oocyte; however, the increase in cleavage rate seen in previous studies after rBST treatment was not observed in the present study. Supplementation of culture medium with rBST during Day 2 to 6 of IVC has been shown to increase the number of trophoblast and subsequent pregnancy rate following transfer. However, in the present study, addition of 50 and 100 ng mL–1 of rBST to the maturation or culture medium did not affect the cleavage rate of embryos and blastocyst production. Table 1.Analysis of the meaning and percentages related to cleavage and production of embryos

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MATURAÇÃO E FECUNDAÇÃO IN VITRO DE OÓCITOS BOVINOS EM TUBOS PREVIAMENTE GASEIFICADOS E MANTIDOS EM BANHO-MARIA

Archives of Veterinary Science ◽

10.5380/avs.v7i2.3991 ◽

2002 ◽

Vol 7 (2) ◽

Author(s):

M. KURTZ FILHO ◽

L. M. SILVA ◽

B. MOREIRA ◽

D. S. BRUM ◽

F. G. LEIVAS ◽

...

Keyword(s):

Water Bath ◽

Bovine Embryo ◽

Bovine Embryos ◽

In Vitro Production ◽

Cleavage Rate ◽

Sodium Pyruvate ◽

Embryo Production ◽

Vitro Production

A produção in vitro (PIV) de embriões bovinos alcançada com vacas de matadouros ou de aspiração folicular in vivo (OPU) é uma prática cada vez mais difundida e a sua simplificação poderia baixar os custos de produção. O objetivo desta pesquisa foi comparar a produção in vitro de embriões em estufa com temperatura, umidade relativa e atmosfera controlada (controle), com tubos de poliestireno gaseificados e mantidos em banho-maria (tratamento). Oócitos obtidos de ovários de vacas abatidas foram maturados in vitro em TCM- 199 modificado com 25mM de N-2-hidroxietilpiperazina-N -2-ácido etanosulfônico (HEPES); 0,025mg/ml de piruvato de sódio, 0,01UI de rFSHh/ml, 0,5µg/ml de LHs e 10% de soro de vaca em estro. Na fecundação in vitro utilizou-se Talp-Fert com 0,06mg/ml de albumina sérica bovina, 0,022mg/ml de piruvato de sódio e 10µg/ml de heparina. O cultivo foi conduzido em placas de 4 poços em SOF com 5% de soro de vaca em estro, 20µl/ml de aminoácidos essenciais e 10µl/ml de aminoácidos não essenciais, sob óleo mineral, em estufa com atmosfera de 5% de CO2, umidade saturada e 39°C, por 9 dias. Na maturação não houve diferença (P>0,05) entre o tratamento e o controle. Porém, a maturação e a fecundação ou somente a fecundação in vitro em tubos mantidos em banho-maria não demonstrou ser uma alternativa recomendada para a produção de embriões bovinos. In vitro maturation and fertilization of bovine oocytes in tubes previously gasified kept in water bath Abstract In vitro bovine embryo production either obtained from oocytes of slaughtered cows or in vivo follicular aspiration (OPU) is a well-known technique and its simplification might reduce the cost of embryo production. The aim of this study was to compare the cleavage rate and embryo development of the in vitro production of bovine embryos using standard culture system (temperature, gas phase and controlled humidity) versus gasified polystyrene tubes kept in water bath. Oocytes obtained from ovaries of slaughtered cows were in vitro maturated in TCM- 199modified with 25 mM of N-2-hidroxyethylpiperazine-N-2-ethanosulfonic acid (HEPES); containing 0.01UI rFSHh/ml and 0.5µg/ml LHs, 0.025mg/ml sodium pyruvate and 10% estrous cow serum. The in vitro fertilization was carried out in Talp-Fert containing 0.06mg/ml BSA, 0.022mg/ml sodium pyruvate and 10µg/ml heparin. The culture was performed in SOF medium with 20µl/ml essential aminoacids, 10µl/ml, non-essential aminoacids and 5% estrous cow serum, with oil overlay, in 4 well dishes and incubated with 5% CO2, maximum humidity at 39°C, for 9 days. The results of this study showed no difference (P>0.05) between the treatment and control groups during the maturation process. However, the maturation and fertilization or only the fertilization in tubes do not represent a viable alternative for the in vitro production of bovine embryos.

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Efficiency of different incubation systems for the in vitro production of bovine embryos

Zygote ◽

10.1017/s0967199418000266 ◽

2018 ◽

Vol 26 (4) ◽

pp. 314-318 ◽

Cited By ~ 1

Author(s):

Camila M. Cavalcanti ◽

Iana S. Campelo ◽

Mirelly M.A.S. Silva ◽

João V.S. Albuquerque ◽

Luciana M. Melo ◽

...

Keyword(s):

Oxygen Species ◽

Bovine Embryos ◽

In Vitro Production ◽

Cleavage Rate ◽

Significant Difference ◽

Maturation Rate ◽

Blastocyst Rate ◽

Vitro Production ◽

Bovine Oocytes

SummaryThis study aimed to compare the efficiency of different incubation systems for in vitro embryo production in bovine. Oocytes/embryos were cultured in three incubators: conventional – CONV, mini bench – MINI and portable – PORT. After in vitro maturation (IVM), oocytes were verified for maturation rate. The remaining structures were submitted to in vitro fertilization and culture to verify cleavage (day 2) and blastocyst (day 7) rates. Reactive oxygen species (ROS) were evaluated in post-IVM oocytes and embryos (days 2 and 7) using arbitrary fluorescence units (AFUs). No significant difference (P>0.05) was observed for maturation rate. The CONV system (74.0%) produced the highest cleavage rate (P<0.05) when compared with PORT (59.5%), but similar (P>0.05) to MINI (65.0%). The same pattern and differences were observed for blastocyst rate: CONV (33.3%), MINI (32.3%) and PORT (21.9%). ROS levels were not different (P>0.05) in post-IVM oocytes: CONV (35.6±4.5), MINI (29.4±4.0) and PORT (35.6±4.5). For day-2 embryos, ROS levels were higher (P<0.05) in MINI (44.2±3.1) in comparison with CONV (27.7±3.7) and PORT (33.3±3.2). No significant difference (P>0.05) was observed in blastocysts. In conclusion, although it produced high ROS levels at day 2 of culture, the MINI system was as efficient as the CONV system for blastocyst production. This option may be an interesting and economical for the in vitro embryo industry.

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