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

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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Use of Melatonin in the In Vitro Production of Bovine Embryos

Revista Brasileira de Saúde e Produção Animal ◽

10.1590/s1519-9940210322020 ◽

2020 ◽

Vol 21 ◽

Author(s):

Alan da Silva LIRA ◽

Ricardo de Macedo CHAVES ◽

Felipe de Jesus MORAES JUNIOR ◽

Sergio Henrique COSTA JUNIOR ◽

Brenda Karine Lima do AMARAL ◽

...

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Control Group ◽

Bovine Embryos ◽

In Vitro Production ◽

Maturation Medium ◽

Significant Difference ◽

Maturation Rate ◽

Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

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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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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

Cercetari Agronomice in Moldova ◽

10.1515/cerce-2017-0040 ◽

2017 ◽

Vol 50 (4) ◽

pp. 109-117

Author(s):

S.I. Borş ◽

Şt. Creangă ◽

D.L. Dascălu ◽

Elena Ruginosu ◽

Mădălina Alexandra Davidescu ◽

...

Keyword(s):

Genetic Gain ◽

Genetic Improvement ◽

Selection Intensity ◽

Bovine Embryos ◽

In Vitro Production ◽

Cleavage Rate ◽

Cattle Breeding ◽

North Eastern ◽

Vitro Production

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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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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292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab292 ◽

2015 ◽

Vol 27 (1) ◽

pp. 235

Author(s):

E. D. Souza ◽

N. C. Rabelo ◽

T. D. Araujo ◽

C. M. Assunção ◽

C. C. R. Quintão ◽

...

Keyword(s):

Heat Shock ◽

In Vitro Maturation ◽

Calf Serum ◽

Developmental Competence ◽

Cleavage Rate ◽

Oocyte Developmental Competence ◽

Significant Difference ◽

Blastocyst Rate ◽

Bovine Oocytes

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

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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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