72 Invitro embryo production using prepubertal calf oocytes with conventional semen and sexed semen ULTRA-4M

2020 ◽  
Vol 32 (2) ◽  
pp. 162
Author(s):  
A. Velazquez ◽  
H. Alvarez ◽  
M. Kjelland ◽  
F. Villaseñor ◽  
G. Ariza ◽  
...  

Invitro embryo production (IVP) can increase the reproductive potential and genetic quality of cattle, as well as other species. This powerful assisted reproduction tool can be used to produce embryos from prepubertal calves, reducing the generation interval. A recent sexed semen technology known as ULTRA (ST Genetics), completely modified the technique, the media and sperm concentration. In field trials with AI there was no difference between conventional semen (CONV) and ultra-sexed semen at a concentration of 4 million per straw (ULTRA-4M). The combined use of IVP and ULTRA-4M can decrease the selection time for improving dairy and beef cattle herd genetics. The objective of this research was to compare the CONV and ULTRA-4M semen using bovine IVP and prepubertal calves. The research was carried out in the reproduction laboratory of the Facultad de Estudios Superiores Cuautitlán - Universidad Nacional Autónoma de México (FESC-UNAM). The IVP was performed with a continuous invitro culture (IVC) system. The ovaries were collected in Campeche, México, from Bos indicus×Bos taurus crossbred calves (6 months old) using surgical castration (for export to the United States) and transported to the laboratory (FESC-UNAM) in BO-HEPES-IVM (Bioscience™), in an oocyte transporter (WTA). Vitrogen media were used for IVF and IVC. For IVM, the cumulus-oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C. Matured oocytes (n=600, divided equally into five replicates) were divided into 2 groups, the CONV group and the ULTRA-4M group. The IVF process was conducted with CONV and ULTRA-4M semen from the same bull (Holstein) at a concentration of 2×106 and 0.5×106 spermatozoamL−1, respectively, for 18h in 38.5°C, 5% CO2, 95% air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The cleavage results were recorded 56h after the beginning of IVC. The cleavage rate, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated. The statistical analysis was carried out with the GLM procedure of the SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of CONV vs. ULTRA-4M (α level=0.05). The percentage of cleavage for CONV was 46%±1.4 and 43.2%±1.4 for ULTRA-4M. The results for embryos with more than 6 cells in the CONV and ULTRA-4M groups were 16%±0.6 and 14%±0.6, respectively. The percentage of blastocysts on Day 7 for CONV was 9%±0.6 and 8%±0.6 for ULTRA-4M. There were no significant differences between groups (P>0.05) for all variables analysed. In conclusion, under the conditions of this research the ULTRA-4M and CONV produced similar results for IVP.

2016 ◽  
Vol 28 (2) ◽  
pp. 215
Author(s):  
S. Castañeda ◽  
S. Romo ◽  
M. E. Kjelland

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.


2020 ◽  
Vol 32 (2) ◽  
pp. 161
Author(s):  
H. Alvarez ◽  
M. Kjelland ◽  
F. Villaseñor ◽  
M. Pérez ◽  
S. Romo

The first commercial production of sexed semen was at the Cogent company in the United Kingdom. Since then millions of offspring have been born using sexed semen produced by flow cytometry. Sexed semen technology has recently been modernized to what is now known as ULTRA (ST Genetics), completely modifying the technique, medium, and sperm concentration. In field trials using AI, there was no difference between conventional semen (CONV) and ultra-sexed semen at a concentration of 4 million sperm per straw (ULTRA-4M). The combination of invitro embryo production (IVP) and sexed semen technologies has been successful for intensity selection in the cattle industry. The objective of this work was to compare the CONV and ULTRA-4M semen for bovine IVP. The research was carried out in the reproduction laboratory at the Facultad de Estudios Superiores Cuautitlán-Universidad Nacional Autónoma de México (FESC-UNAM). The IVP was performed with a continuous invitro culture (IVC) system. Ovaries (n=213) were collected from a slaughterhouse (Querétaro, México) and transported to the laboratory within 2h (FESC-UNAM) in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IUmL−1) and streptomycin sulfate (100µgmL−1). For IVP, VITROGEN media were used for IVM, IVF, and IVC. For the IVM, the cumulus-oocyte complexes were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2, 95% air, and 100% humidity. Matured oocytes (n=1000) were divided into two groups, the CONV group and the ULTRA-4M group. The IVF process was developed with CONV and ULTRA-4M semen from the same bull (Holstein) at a concentration of 2×106 and 0.5×106 spermmL−1, respectively, for 18h in 38.5°C, 5% CO2, 95% air, and 100% humidity. The presumptive zygotes were denuded by pipetting and left in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The cleavage rate, embryos of more than 6 cells, and blastocysts on Day 7 of culture were evaluated. The statistical analysis was carried out with the GLM procedure of the SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of CONV vs. ULTRA-4M (α level=0.05). Percent cleavage for CONV was 72.2%±2.53 and 75.6%±2.53 for ULTRA-4M. For embryos with more than 6 cells, the results for CONV and ULTRA-4M were 59.8%±5.61 and 62.8%±5.61, respectively. The percentage of blastocysts on Day 7 was 37.8%±5.39 for CONV and 43.6%±5.39 for ULTRA-4M. There were no significant differences between the groups (P>0.05) for all variables analysed. Although the number of blastocysts on Day 7 were numerically higher in the ULTRA-4M, differences were not significant. In conclusion, under the conditions of this research the ULTRA-4M had similar results as CONV for bovine IVP.


2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 37-37
Author(s):  
Nicola Oosthuizen ◽  
Pedro Levy Piza Fontes ◽  
G Cliff Lamb

Abstract Bos indicus and Bos taurus cattle diverged from an evolutionary standpoint more than 110,000 years ago. Since then, Bos indicus cattle have undergone genetic adaptations beyond the commonly discussed increased thermo-tolerance and parasite resistance. Several physiological differences exist between Bos indicus and Bos taurus cattle, and it is important to consider these differences when establishing reproductive management strategies. It has been well described that Bos indicus cattle have a delayed onset of puberty and longer periods of postpartum anestrus, yet through the utilization of estrus synchronization protocols these challenges can be attenuated. However, when Bos indicus females are exposed to these protocols, they are known to have smaller dominant follicles, lower expression of estrus, and decreased pregnancy rates to artificial insemination (AI) when compared to Bos taurus females. These factors can be overcome through the utilization of estradiol and progesterone based synchronization protocols, which improve follicular dynamics and yield acceptable pregnancy rates to assisted reproductive technologies in cattle adapted to tropical or subtropical conditions. However, the use of estrogens for synchronization purposes is not permitted in the United States, and cattle producers need to rely on GnRH-based protocols. Another key difference between subspecies, is that Bos indicus females have greater antral follicle counts than Bos taurus females, which proves beneficial for in vitro embryo production. Therefore, an opportunity exists to explore the greater productivity of donors in embryo production in order to improve genetics in herds that utilize these breeds.


2015 ◽  
Vol 156 ◽  
pp. 58-63 ◽  
Author(s):  
William Omar Contreras Lopez ◽  
Hernando Raphael Alvis-Miranda ◽  
Andres Felipe Gamarra ◽  
Bibiana Rendon ◽  
Diego Armando Vega Borda ◽  
...  

2019 ◽  
Vol 51 (8) ◽  
pp. 2641-2644
Author(s):  
Fernando Naranjo-Chacón ◽  
Felipe Montiel-Palacios ◽  
Rodolfo Canseco-Sedano ◽  
Concepción Ahuja-Aguirre

2016 ◽  
Vol 28 (2) ◽  
pp. 183
Author(s):  
S. J. R. Rodriguez ◽  
Y. E. Ramirez ◽  
E. Gomes ◽  
L. F. Nasser ◽  
J. H. F. Pontes ◽  
...  

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.


2010 ◽  
Vol 22 (1) ◽  
pp. 293 ◽  
Author(s):  
L. U. Gimenes ◽  
M. L. Ferraz ◽  
A. Araujo ◽  
P. Fantinato Neto ◽  
M. R. Chiarati ◽  
...  

One important factor in the success of ovum pickup (OPU)/IVP in Bos taurus is the follicular status at OPU concerning the dominance period (Hendriksen et al. 2000 Theriogenology 53, 11-20). The hypothesis of the present study is that OPU performed after follicle deviation, when follicles show a mild level of atresia, improves competence for IVP in Nelore (NE), Holstein (HO), and buffaloes (BU). Objectives were to determine effects of OPU done at different times of synchronized follicular wave (1, 3, or 5 d after expected emergence) and of genetic group (NE, HO, and BU) on IVP. A total of 27 heifers (9 of each genetic group) were maintained in contemporary nutritional and environmental conditions during experiment, in a cross-over design, performed in 6 replicates. Recovered oocytes with at least one cumulus cell layer were matured in TCM-199 supplemented with 10% of FCS plus 50 μM of cysteamin and 0.3 mM of cystine, at 38.5°C with 5% CO2 in air for 24 h. IVF was done with 2 × 106 spermatozoa per mL of NE (for bovine oocytes) or BU semen (for BU oocytes), for 20 h at the same incubator conditions of IVM. After IVF, presumptive zygotes were denuded and cultured in SOF under the same previous atmosphere conditions. Medium was changed 3 d after IVF when cleavage rate (CR) was assessed. Blastocyst (BR) and hatching rates (HR) were evaluated 7 and 9 days after IVF, respectively. About 50% of hatched blastocysts were fixed until nuclei counting. Data were analyzed by ANOVA using the Proc Mixed model. No effects of interaction or time of synchronization were observed in any of the variables. Concerning genetic group, NE had better results than HO and BU (mean ± SEM / heifer / replicate), respectively, for visualized follicles (41.0a ± 2.1, 22.1b ± 1.3, 18.8b ± 0.9), total oocytes (37.1a ± 2.5, 15.4b ± 1.2, 14.8b ± 1.0), oocytes at IVM (30.8a ± 2.4, 10.7b ± 1.0, 7.9b ± 0.7), oocytes at IVC (18.7a ± 0.8, 8.0b ± 0.5, 7.5b ± 0.4), cleaved embryos (15.4a ± 0.7, 4.6b ± 0.4, 4.4b ± 0.3),CR(81.8a, 59.1b, 62.3b), blastocysts on Day 7 (5.1a ± 0.6, 1.0b ± 0.2, 0.6b ± 0.1), BR (25.8a, 13.6b, 9.1b), and hatched blastocysts on Day 9 (2.6a ± 0.4, 0.3b ± 0.1, 0.3b ± 0.1). Recovery rate and HR were greater for NE (89.4 and 50.6%, respectively) than for HO (73.3 and 23.2%), but neither differed from BU (82.8 and 31.9%). Also, the percentage of viable was greater for NE (83.0) than for HO (66.9) and BU (53.1). No effects were observed for nuclei counting (NE = 176.6 ± 5.3, HO = 168.9 ± 13.7 and BU = 206.1 ± 23.1). Results demonstrate that Nelore had a better efficiency for IVP than Holstein and buffaloes. OPU performed at different times of synchronized follicular wave did not influence IVP, conversely to the initial hypothesis of this study. FAPESP (06/59550-6, 07/04782-2), Tortuga Cia Zootecnica®, Santa Adele and São Caetano Farms, LMMD, PCAPS, HOVET (Dr. Ubiraem Schalch), VRA, VNP (Prof. Dr. Francisco de Palma Rennó).


2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.


2010 ◽  
Vol 22 (1) ◽  
pp. 248 ◽  
Author(s):  
J. H. F. Pontes ◽  
K. C. F. Silva ◽  
A. C. Basso ◽  
C. R. Ferreira ◽  
G. M. G. Santos ◽  
...  

In recent years, Brazil has become the leading country in the world for the number of embryos produced in vitro (Thibier M 2009 IETS Embryo Transfer Newsletter 22, 12-19). This is partly due to the large numbers of Bos indicus animals in Brazil, making up about 80% of the total cattle. The mean oocyte production per ultrasound-guided follicular aspiration from Bos indicus is higher than those for European breeds (Pontes JHF et al. 2009 Theriogenology 71, 690-697). In the present study, we analyzed 5407 ovum pick ups (OPU) and compared the average production of total (n = 90,086) and viable (n = 64,826) oocytes and the number of embryos produced in vitro from Gir (Bos taurus indicus), Holstein (Bos taurus taurus), 1/4 Holstein × 3/4 Gir, and 1/2 Holstein-Gir crossbreed cows. To obtain oocytes, OPU was repeated from 4 to 7 times (mean = 5.7 ± 2.4) in each donor cow aged from 3 to 7 years (mean = 5.0 ± 2.3) during a 12-mo period. COCs (n = 90,086) obtained were classified according to the presence of cumulus cells and the oocyte cytoplasm aspect (homogeneous or heterogeneous/fragmented). The viable oocytes (n = 64,826) were in vitro matured for 24 h at 38.8°C in an atmosphere of 5% CO2 in air. Since this was a commercial programm, frozen sexed semen (2 × 106 mL-1) from Gir (n = 8) or Holstein (n = 7) sires previously tested for high efficiency was used for IVF. Fertilization was carried out (18-20 h) and the presumed embryos were cultured for 7 days in the same conditions as were used for IVM. Data were analyzed by ANOVA. On average, 16.7 ± 6.2 oocytes were obtained per OPU/IVF procedure and 71.96% were considered viable. The mean numbers of total oocytes per OPU/IVF procedure were 17.1 ± 4.4 for Gir cows (n = 617), 11.4 ± 3.9 for Holstein cows (n = 180), 20.4 ± 5.8 for 1/4 Holstein × 3/4 Gir (n = 44), and 31.4 ± 5.6 for 1/2 Holstein-Gir crossbreed females (n = 37, P < 0.01). The mean numbers of viable oocytes per OPU/IVF procedure were 12.1 ± 3.8 for Gir cows, 8.0 ± 2.6 for Holstein cows, 16.8, ± 5.0 for 1/4 Holstein × 3/4 Gir, and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbreed females (P < 0.01). The average number of embryos produced by OPU/IVF were 3.2 (n = 12,243/3378) for Gir cows, 2.2 (n = 2426/1138) for Holstein cows, 3.9 (n = 1033/267) for 1/4 Holstein × 3/4 Gir, and 5.5 (n = 1222/224) for 1/2 Holstein-Gir. The average number of embryos produced per IVF session from 1/2 taurus × indicus donor cows was greater (P < 0.01) than from Bos indicus cows. The number of recoverable and viable oocytes and the number of embryos produced in vitro from Bos indicus donors were higher than from Bos taurus females. Therefore, the highest oocyte yield and the greatest embryo production were obtained from 1/2 taurus × indicus females. This work was supported by In Vitro Brasil.


2015 ◽  
Vol 98 (5) ◽  
pp. 3086-3099 ◽  
Author(s):  
J.N.S. Sales ◽  
L.T. Iguma ◽  
R.I.T.P. Batista ◽  
C.C.R. Quintão ◽  
M.A.S. Gama ◽  
...  

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