102 In Vitro Embryo Production and Oocyte Quality in Bos indicus Beef Cows Selected for Fertility Characteristics

Embryo production may be enhanced when associated with cows selected on the basis of fertility markers, which should be easy to measure, such as antral follicle count (AFC) and genital tract morphometrics. The objective was to evaluate the effects of AFC class on oocyte 24-h outcome and in vitro embryo production in Bos indicus beef cows. Brahman (n = 151) cows (2-13 years old, 344-803 kg of BW, and 7-9 BCS). Low (LAFC), intermediate (IAFC), and high (HAFC) antral follicle classes were defined as follows: LAFC ≤ 30; IAFC 30-49; and HAFC ≥50 AFC. All follicles ≥3 mm in diameter were aspirated by conventional ovum pick-up technique. Only cumulus–oocyte complexes with at least 2 layers of granulosa cells and homogeneous cytoplasm were used for in vitro culture. They were matured in TCM-199 plus supplements for 24 h at 38.7°C in a 5% CO2 humidified atmosphere. After 24 h of maturation, a subset of oocytes (n = 319) was fixed and analysed under fluorescent microscopy and oocyte outcome was evaluated by classification, as follows: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I/anaphase I/telophase I (MIAITI), and metaphase II (MII). The second subset of oocytes (n = 797) was fertilized in Ferti-TALP (10-15 oocytes per 60-µL drop) with frozen–thawed semen (18-22 h at 38.7°C in 5% CO2 after Percoll) from a single bull previously tested for good in vitro fertility. Presumptive zygotes were cultivated in CR2 medium for 48 h at 37.8°C in 5% CO2. For the remaining 96 h, embryos were transferred to 10% FCS-supplemented TCM-199 drops until the final evaluation. Data were analysed by the GENMOD, GLM, and CORR procedures of SAS (SAS Institute Inc., Cary, NC, USA). Viable oocytes, total embryos, and embryo production efficiency (viable oocyte/total embryos produced; P < 0.05) to AFC in various degrees (r2 = 0.87, 0.86, 0.30, respectively). The proportion of oocytes in GV, GVBD, MIAITI, and MII were different (P < 0.05) between LAFC, IAFC, and HAFC classes [GV: 12.3% (13/106)a, 3.1% (3/96)a and 4.3% (5/117)b, respectively]; [GVBD: 32.1% (34/106)a, 8.3% (8/96)a and 6.0 (7/117)b]; [MIAITI: 14.2% (15/106)a, 26.0% (25/96)b and 8.5% (10/117)c, respectively] and [MII: 41.5% (44/106)b, 62.5% (60/96)a and 81.2% (95/117)c, respectively). In conclusion, high AFC is positively related to better in vitro embryo fertility and to 24-h oocyte outcome after in vitro maturation.

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169 OVUM PICK-UP, IN VITRO EMBRYO PRODUCTION, AND EMBRYO TRANSFER IN CATTLE IN TROPICAL AND SUBTROPICAL MEXICO

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab169 ◽

2016 ◽

Vol 28 (2) ◽

pp. 215

Author(s):

S. Castañeda ◽

S. Romo ◽

M. E. Kjelland

Keyword(s):

Embryo Transfer ◽

Production Efficiency ◽

Bos Taurus ◽

Bos Indicus ◽

Hormonal Treatment ◽

Cleavage Rate ◽

Embryo Production ◽

Sexed Semen ◽

In Vitro Embryo Production

Biotechnology continues to evolve rapidly, allowing the development of artificial reproductive techniques (ART) to increase reproductive efficiency and contribute to the genetic improvement of domestic animals. The present study examines the results obtained after 30 months of starting a commercial practice for ovum pickup (OPU) in vitro embryo production (IVP) and embryo transfer (ET) in cattle in tropical and subtropical Mexico. This research was conducted from 2013–2015 in beef and dairy cattle kept under different environmental and management conditions in 6 states (Chiapas, Oaxaca, Tabasco, Tamaulipas, Veracruz, and Yucatan). Oocytes were collected by OPU, without hormonal treatment, from 10 donor cows: 2 Bos taurus (Bt), 5 Bos indicus (Bi), and 3 Bt × Bi. A total of 98 oocyte recovery sessions were performed on 756 cows and produced 12 524 viable oocytes (1349 GI, 3383 GII, 7792 GIII), which were sent to a central laboratory for IVP. Both conventional and sexed semen were used for IVF, from 9 breeds: 2 Bt, 5 Bi and 2 Bt × Bi. The overall cleavage rate was 69% (8587/12 524). The embryo production efficiency rate was 31% (3905/12 524). Fresh sexed and conventional embryos were transferred to recipients synchronised with the following protocol: Day 0, application of an intravaginal device (ID) with progesterone and 2 mg of oestradiol benzoate IM; Day 8, removal of the ID, 400 IU eCG IM, 0.5 mg cloprostenol sodium IM, and 0.5 mg oestradiol cipionate IM. Day 10 was considered the day of oestrus. Pregnancy rate after ET was 38% (945/3905). The average number of viable oocytes per donor cow was 16.57; the average number of transferred embryos per donor cow was 5.17, and the average number of pregnancies per donor cow was 1.25. The OPU-IVP were successful in producing pregnancies even under several adverse conditions, such as a tropical environment, many donors being prepuberal females and the majority of the adult cows having a previous non-productive history due to hormonal mishandling in superovulation programs or because of overfeeding for show purposes. The consequences of these factors can be observed in the lower overall cleavage rate obtained (69%), compared to the expected (75%). Some variables require further analysis (e.g. different OPU and ET technicians, time of year, cow age, cow breed, and use of conventional and sexed semen). A successful IVP practice has to face and overcome multiple problems that may arise in some geographic locations but, perhaps, not in others. It is interesting to note the use of sexed versus conventional embryos, of the 98 ET sessions, 25 involved sexed embryos versus 73 for conventional embryos. The use of sexed semen for OPU-IVP and ET in this region of Mexico continues to expand. We thank Genemex Internacional and the ranch owners that were involved with this research.

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106 COMPARISON OF IN VITRO EMBRYO PRODUCTION IN PANAMA USING HOLSTEIN OOCYTES WITH BOS INDICUS SEXED SEMEN FROM DONORS IN DIFFERENT LOCATIONS: FIELD DATA

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab106 ◽

2016 ◽

Vol 28 (2) ◽

pp. 183

Author(s):

S. J. R. Rodriguez ◽

Y. E. Ramirez ◽

E. Gomes ◽

L. F. Nasser ◽

J. H. F. Pontes ◽

...

Keyword(s):

Embryo Development ◽

Bos Indicus ◽

Embryo Production ◽

University Of Illinois ◽

Oocyte Collection ◽

Sexed Semen ◽

The Usa ◽

In Vitro Embryo Production ◽

The University

The objective of this work was to compare in vitro embryo production of Bos taurus × Bos indicus cross embryos using oocytes from Holstein donors under different production and environment systems. This study also examined the possibility for in vitro production using oocytes imported and transported fresh between the USA and Panama. All animals were mature Holstein cows going through a normal lactation. The first group of donors was from the University of Illinois dairy herd and went through 3 ovum pickup sessions. The second group of donors were Holstein cows already adapted to Panama and went through 10 ovum pickup sessions. The Panamanian herd of Holstein donors were born and raised in Panama in an area of mountains, on average 1300 m above sea level. This environment does not have the typical hot and humid tropical weather seen in other regions of Panama. Both groups of donors were aspirated without stimulation during the years 2013 and 2014. Oocytes recovered from donors in Illinois were imported fresh under a special sanitary research protocol between Panama and the University of Illinois. The transport of fresh oocytes from the USA to Panama was done using a portable incubator set at 39°C (Minutube of America). Oocytes were matured during transport in 5-mL tubes (~30–35 oocytes per tube) containing 400 µL of maturation media (TCM-199) that had been equilibrated with 5% CO2. Oocytes recovered from donors in Panama were matured using the same media. For both groups, oocytes were inseminated 24 h after ovum pickup using sexed semen from the same bull. All embryo production procedures followed the protocols of the In vitro Brasil™ commercial system. At 72 h postinsemination, cleavage was evaluated. On Day 7 after insemination, embryo development to the blastocyst stage (early to expanded) was recorded. Data were analysed using Chi-squared. As shown in Table 1, there was no effect of oocyte collection location on embryo development. These results indicate that it is possible to produce a viable in vitro-produced embryo using fresh oocytes collected and transported from different countries. This work opens the possibility to access superior genetics and improve herds in countries seeking to increase their production systems and potentially improve their quality of life. Table 1.Effect of oocyte collection location on embryo development This project was supported by Programa de Competitividad ProCom Senacyt, Panama.

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281 EVALUATION OF IN VITRO EMBRYO PRODUCTION EFFICIENCY USING SEXED BULL SPERM SORTED WITH TWO TYPES OF CELL SORTER

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab281 ◽

2011 ◽

Vol 23 (1) ◽

pp. 238

Author(s):

H. Hayakawa ◽

T.-I. Hirata

Keyword(s):

Production Efficiency ◽

Bos Taurus ◽

Digital Processing ◽

Embryo Production ◽

Cell Sorter ◽

Treatment Groups ◽

Fort Collins ◽

In Vitro Embryo Production ◽

Bull Sperm

Cell sorting is an important part of the sperm sexing process. The objective of this study was to compare the efficiency of in vitro embryo production using sexed frozen–thawed bull sperm sorted with 2 types of cell sorter. Ejaculates from 2 Bos taurus (Holstein, 5 years old) bulls underwent conventional processing (control) or sorting for X chromosome bearing sperm using MoFlo® SX (SX, Dako, Fort Collins, CO, USA) or MoFlo® XDP-SX (XDP, Beckman Coulter, Fullerton, CA, USA) following XY™ sperm-sorting protocols. Processed sperm samples were cryopreserved in 0.5-mL plastic straws. Cumulus–oocyte complexes obtained from abattoir-derived ovaries were matured for 20 h in HEPES–TCM-199 (Lu and Seidel 2004 Theriogenology 62, 819–830) and randomly assigned to each of 3 sperm treatment groups. Thawed sperm were centrifuged for 20 min at 448 × g through an ISolate® (Irvine Scientific, Santa Ana, CA, USA) gradient (45:90%). Sperm pellets were washed in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) by centrifugation for 5 min at 252 × g. Oocytes were co-incubated with washed sperm (5 to 10 × 106 sperm mL–1) in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) for 8 h at 38.5°C in 5% CO2 and 95% air (Day 0). Presumptive zygotes were cultured for 90 h in CDM-1 (Lu and Seidel 2004 Theriogenology 62, 819–830) and then washed and cultured in IVD101 (Hoshi 2003 Theriogenology 59, 675–685) at 38.5°C in 5% CO2, 5% O2, and 90% N2. Cleavage rates on Day 2 and blastocyst rates on Day 7 to 9 were recorded after insemination. Two-way ANOVA was used for data analysis, followed by Fisher’s PLSD test. Experiments were replicated 4 times for bull A (total of 1 350 oocytes used) and 5 times for bull B (total of 1 529 oocytes used). The data are summarised in Table 1. No interaction was observed between the treatments and bulls. Cleavage rates were not significantly different in the 3 treatment groups. However, blastocyst rates were significantly lower in both SX (P < 0.001) and XDP (P < 0.002) groups than in control groups for both bulls but not different between SX and XDP (P > 0.8). Bull B showed significantly poorer results than bull A regarding both cleavage (P < 0.003) and blastocyst (P < 0.02) rates. MoFlo® SX (analogue processing) has been used for a decade, and XDP (digital processing) is the replacement model with its accelerated sorting speed. The current results indicated that the in vitro embryo production efficiency did not differ between sperm sorted with either SX or XDP. We suggest that sperm can be sorted using XDP without compromising sperm health. Table 1.Cleavage and blastocyst rates after IVF with 2 Holstein bulls for three sperm treatments

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Effects of a high-energy diet on oocyte quality and in vitro embryo production in Bos indicus and Bos taurus cows

Journal of Dairy Science ◽

10.3168/jds.2014-8858 ◽

2015 ◽

Vol 98 (5) ◽

pp. 3086-3099 ◽

Cited By ~ 24

Author(s):

J.N.S. Sales ◽

L.T. Iguma ◽

R.I.T.P. Batista ◽

C.C.R. Quintão ◽

M.A.S. Gama ◽

...

Keyword(s):

Bos Taurus ◽

Bos Indicus ◽

Oocyte Quality ◽

High Energy ◽

Embryo Production ◽

In Vitro Embryo Production

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Supplementation of c-type natriuretic peptide during the whole in vitro growth period benefits the development of murine secondary follicles

10.21203/rs.3.rs-16148/v1 ◽

2020 ◽

Author(s):

Ang Li ◽

Haixia Cao ◽

Hongxia Li ◽

Ruijiao Li ◽

Huaixiu Wang ◽

...

Keyword(s):

Natriuretic Peptide ◽

Ovulation Induction ◽

Germinal Vesicle ◽

Antral Follicle ◽

Control Group ◽

Follicle Development ◽

In Vitro Growth ◽

Germinal Vesicle Breakdown ◽

Preovulatory Follicles

Abstract Background Supplementation of c-type natriuretic peptide (CNP) in the culture medium shortly before in vitro maturation (IVM) has been reported to be effective in delaying meiotic resumption of murine oocyte. The present study investigated the effect of CNP supplementation during the whole period of in vitro growth (IVG) on the development of murine secondary ovarian follicles.Methods Late secondary ovarian follicles isolated from ovaries of Kunming mice were cultured in vitro with and without supplementation of CNP. In experiment 1, CNP was supplemented at the early stage and the follicle development was evaluated. In experiment 2 and 3, CNP was supplemented during the whole period of IVG. In experiment 2, follicle development and oocyte maturity were evaluated. In group 3, follicle development and rate of cleaved embryos after in vitro fertilization (IVF) was assessed.Results In control group in all 3 experiments, granulosa cells migrated from within follicle and adhered to the plate at different degrees. The follicles flattened and could not reach antral stage. About 39.8% (39/98) of the oocytes ovulated nakedly. As no antral follicle was obtained, IVF was not performed in control group in experiment 3. In experiment group in all 3 experiments, no migration of guanulosa cells was observed and the follicles grew three-dimensionally. Ovulation of naked oocyte decreased substantially. The rate of antral stage follicle were 45% (18/40) in experiment 1. This parameter was 75.9% (44/58) in experiment 2 and 3 combined. In experiment 2, in preovulatory follicles without ovulation induction, oocytes at germinal vesicle (GV) stage and germinal vesicle breakdown (GVBD) stage were 87.5% (14/16) and 12.5% (2/16), respectively. In preovulatory follicles with ovulation induction, no GV stage oocyte was retrieved, oocytes at GVBD and metaphase II (MII) stage were 50% (8/16), respectively. In experiment 3, among 18 follicles cultured, 12 cumulus-oocyte complexes (COC) ovulated automatically after ovulation induction. Eleven oocytes were fertilized and cleaved. Compared with control groups, the follicle development assessed by naked oocyte ovulation and follicle stage (preantral follicle and antral follicle) in experiment groups were significantly superior (p<0.0001). CNP effectively maintained oocytes’ meiotic arrest and enhanced fertilization competency.Conclusions The supplementation of CNP in culture system of murine late secondary follicle during the whole period of IVG could sustain the 3-dimensional structure of follicle, increase the antral formation rate. As a result, the oocyte’s competency to be fertilized was greatly improved.

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Effect of different cryopreservation extenders added with antioxidants on semen quality and in vitro embryo production efficiency in cattle

Anais da Academia Brasileira de Ciências ◽

10.1590/0001-3765202120191229 ◽

2021 ◽

Vol 93 (3) ◽

Author(s):

NATALIA C. SILVA ◽

KAREN M. LEÃO ◽

JOÃO T. PÁDUA ◽

THAISA C. MARQUES ◽

FRANCISCO R.A. NETO ◽

...

Keyword(s):

Production Efficiency ◽

Semen Quality ◽

Embryo Production ◽

In Vitro Embryo Production

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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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