262 REPORTS OF IN VITRO PRODUCTION AND PREGNANCY RATES OF BOVINE EMBRYOS PRODUCED BY POST-THAWING SEXED 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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241 EFFECT OF PRODUCTION EFFICIENCY IN THE LIKELIHOOD OF PREGNANCY OF IN VITRO-DERIVED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab241 ◽

2015 ◽

Vol 27 (1) ◽

pp. 210

Author(s):

L. F. Feres ◽

L. S. A. Camargo ◽

M. P. Palhao ◽

F. Z. Brandao ◽

J. H. M. Viana

Keyword(s):

Pregnancy Rate ◽

Production Efficiency ◽

Production Systems ◽

Bos Indicus ◽

Pregnancy Rates ◽

In Vitro Production ◽

Cleavage Rate ◽

Embryo Production ◽

Chi Square

Improving in vitro culture systems to optimize embryo yield has been a major research goal. The relationship between the efficiency of embryo production systems and the pregnancy outcomes, however, remain controversial. The aim of the present study was to evaluate the likelihood of pregnancy of in vitro-produced embryos derived from batches with different relative efficiency indexes. Data of 702 ovum pick-up (OPU) and in vitro embryo production (IVEP) sessions, and of 2456 embryo transfers, recorded from 2008 to 2012, were evaluated. All donors were from the same herd, and were of the same breed (Gir, Bos indicus), as well as the semen used for IVF. The cumulus-oocycte complex (COC) recovery and IVEP were performed by the same team, in a single IVF laboratory, and using standard medium and procedures. Only data from embryos transferred as fresh were used, and records from 97 OPU/IVEP sessions in which no embryo was produced, or embryos were frozen or discharged due to lack of recipients, were discharged. The remaining 605 sessions were stratified in quartiles (I to IV, each one corresponding to 25% of total data) according to COC production of the donors, or stratified in ranges (0–25%, 26–50%, 51–75%, and 76–100%) according to COC quality (percentage of viable COC or of grade I COC) and to embryo production efficiency endpoints (cleavage rate, blastocyst rate). Pregnancy rates were compared among quartiles or ranges by the chi-square method. On average, the Gir donors produced 24.8 ± 0.6 COC per OPU, from which 14.4 ± 0.4 were classified as viable (57.8%), and 3.2 ± 0.1 as grade I (12.9%). On average 6.1 ± 0.2 embryos (morulas and blastocysts) were produced per OPU per donor, and mean pregnancy rate was 30.9%. As expected, donors with greater total COC yield (quartile I) also produced more viable oocytes (25.5 ± 0.7 v. 15.7 ± 0.3, 10.5 ± 0.2 and 5.8 ± 0.2), more COC grade I (4.8 ± 0.4 v. 3.9 ± 0.3, 2.6 ± 0.2 and 1.6 ± 0.1), and more embryos (9.0 ± 0.4 v. 6.9 ± 0.3, 5.0 ± 0.2 and 3.3 ± 0.1) than donors from quartiles II, III, or IV, respectively (P < 0.0001). Nevertheless, there was no difference (P > 0.05) in pregnancy rates for embryos produced from donors ranked in the different quartiles (30.9 v. 29.3, 31.5, and 30.5% for quartiles I to IV, respectively). Similarly, there was no difference (P > 0.05) in the pregnancy rate of embryos derived from OPU sessions in which there was a high or low percentage of viable or grade I COC. In vitro production efficiency (cleavage and blastocyst rates) also had no effect (P > 0.05) on further pregnancy rates. In conclusion, these results suggest that there is no relationship among the average number or quality of the COC recovered by OPU, the efficiency of IVEP, and the likelihood of pregnancy of in vitro-derived embryos.Research was supported by Fazendas do Basa, CNPq, and Fapemig.

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144 PREGNANCY RATES FOLLOWING THE TRANSFER OF IN VITRO FERTILIZED EMBRYOS TO RECIPIENTS ON DAY 6, 7, OR 8 AFTER OESTRUS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab144 ◽

2015 ◽

Vol 27 (1) ◽

pp. 164

Author(s):

R. C. Fry ◽

K. L. Fry ◽

H. A. McCartney ◽

W. R. Geddes ◽

K. Geddes

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Pregnancy Rates ◽

Transfer Program ◽

In Vitro Production ◽

Chi Squared ◽

Grade 3 ◽

Vitro Production ◽

Embryo Grade

The aim of this experiment was to investigate the effect of day of synchrony on the pregnancy rate of recipients following the transfer of Day 7 IVF embryos. In addition, the effect of IVF embryo grade and corpus luteum (CL) grade of recipients was determined. A total of 317 cumulus-oocyte complexes collected from 24 dry Brahman cows by TVR were matured, fertilized, and cultured under standard in vitro production procedures (Fry et al. 2003 Theriogenology 59, 446). A total of 89 (44 Grade 1, 43 Grade 2, and 2 Grade 3, IETS classification) in vitro-produced embryos were transferred to parous 4- to 9-year-old dry Brahman cross recipient cattle 7 days after IVF. Two groups of recipient cows were synchronised one day apart with an 8-day CIDR/pg protocol so that oestrous would be concentrated over 3 days with the middle day aligning with the day of IVF (Day 0). Donors that produced a large number of IVF embryos had these divided and transferred into recipients either on Day –1 or Day +1 of synchrony, and those producing less than 4 IVF embryos were transferred into recipients on Day 0. At embryo transfer the ovaries of the recipient were palpated and then scanned by rectal ultrasound and the grade of CL noted (Grade 1 = large distinct CL by palpation, Grade 2 = small distinct CL by palpation, Grade 3 = CL not distinguishable by palpation). Pregnancy was diagnosed by ultrasound scanning on Day 92. Although recipient numbers were low, differences in pregnancy rate between groups were analysed by Chi-squared. Data from the 2 Grade 3 embryos transferred were not included in the analysis (0/2 pregnant). Similar (P > 0.05) pregnancy rates were found when Day 7 IVF embryos were transferred to either Day 6 (17/32 = 53%), Day 7 (9/24 = 38%), or Day 8 (14/31 = 45%) recipients. Furthermore, neither the grade of the embryo (Grade 1: 20/44 = 45%, Grade 2: 20/43 = 47%) nor the grade of recipient CL (Grade 1: 17/45 = 38%, Grade 2: 17/29 = 59%, Grade 3: 6/13 = 46%) effected pregnancy rate (P > 0.05). This experiment demonstrates the flexibility of the IVF embryo to achieve an acceptable pregnancy rate over a range of recipient stages thereby allowing a high usage rate of good-quality recipients in an IVF embryo transfer program.

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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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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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