scholarly journals GnRH or estradiol benzoate combination with CIDR improves in-vivo embryo production in bovines (Bos indicus and Bos taurus) under subtropics

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12077
Author(s):  
Khalid Mahmood ◽  
Muhammad Zahid Tahir ◽  
Mahboob Ahmad Butt ◽  
Shazia Mansoor Qureshi ◽  
Amjad Riaz
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
Estradiol Benzoate ◽  
Limiting Factors ◽  
Control Group ◽  
Corpora Lutea ◽  
Embryo Production ◽  
Varied Response ◽  
Donor Cows

Multiple Ovulation and Embryo Transfer (MOET) technology is a potential technique to upgrade livestock species’ genetics. The varied response to super-stimulatory treatments remains one of the limiting factors to this technology’s widespread use. The present study was aimed to improve the superovulation response and in-vivo embryo production by using controlled internal drug release (CIDR)-GnRH or CIDR-EB (Estradiol Benzoate) along with conventional superovulation protocol in Holstein Frisian (HF): Bos taurus; n = 42) and Crossbred (XB: Cholistani (Bos indicus) × HF; n = 28) cows. In the CIDR-GnRH/CIDR-EB treatment, CIDR was implanted in the cows after confirming the presence of a corpus luteum (CL) on the 8th day after estrus. 2 ml GnRH (Lecirelin acetate 0.0262 mg/ml) or 2 mg EB was also administered in CIDR-GnRH/CIDR-EB groups, respectively. Both groups were given super-stimulatory treatment from the 11th day after estrus (FSH in tapering doses twice a day for four consecutive days). On day 13, two doses of 2 ml prostaglandin (75 µg/ml of dextrorotatory cloprostenol) were administered (am: pm), and CIDR was removed the following day. Two artificial inseminations (AI) of the cows were performed (12 h apart) on the 15th day. No CIDR and GnRH/E.B were given in the control group, but the remaining superovulation protocol was the same. Later on, seven days after the first AI, non-surgical embryo flushing was done. The transferable embryos produced from three different superovulation protocols were then transferred into the recipient cows (n = 90) for determining their fertility. Statistical analysis revealed that the number of super-estrus follicles (SEF), multiple corpora lutea (MCL), ovulation/fertilization percentage, fertilized structures recovered (FSR), and transferable embryos (TEs) remained significantly higher (p < 0.05), and days taken for return to estrus (RTE) after embryo collection remained significantly lower (p < 0.05) in CIDR-GnRH (n = 18) and CIDR-EB (n = 15) groups as compared to the control (n = 37). The comparison between XB and HF cows revealed that the TEs production in CIDR-GnRH (XB = 5 vs HF = 13) and CIDR-EB (XB = 6 vs HF = 9) based superovulation protocols were 11.60  ±  4.08 vs 04.31  ±  0.98 and 09.33  ±  1.78 vs 05.22  ±  1.36, respectively. TEs production in XB cows (n = 5) of the CIDR-GnRH group was significantly higher (11.60  ±  4.08) than other groups. On the other hand, the days taken for RTE after embryo collection remained significantly lower (p < 0.05) in HF cows of treatment groups. However, the fertility of TEs was neither affected significantly (p > 0.05) by the superovulation protocol used nor by breed differences among donor cows. In conclusion, using CIDR-GnRH or CIDR-EB along with conventional superovulation protocol may enhance the efficiency of MOET programs in cattle. Furthermore, XB donor cows demonstrated a better performance than HF donor cows under subtropical conditions.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

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.

179 COMPARISON OF OOCYTE AND EMBRYO PRODUCTION AMONG BOS TAURUS, BOS INDICUS, AND INDICUS-TAURUS DONOR COWS

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 ◽  
...  
Keyword(s):  
High Efficiency ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Cumulus Cells ◽  
Holstein Cows ◽  
Embryo Production ◽  
Total N ◽  
The Mean ◽  
Donor Cows

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.

225 ADDITION OF EQUINE CHORIONIC GONADOTROPIN TO A TRADITIONAL FOLLICLE STIMULATING HORMONE PROTOCOL FOR SUPEROVULATION OF BOS TAURUS BEEF COWS

2012 ◽  
Vol 24 (1) ◽  
pp. 224 ◽  
Author(s):  
R. L. Davis ◽  
A. Arteaga ◽  
J. F. Hasler
Keyword(s):  
Treatment Group ◽  
South America ◽  
Bos Taurus ◽  
Animal Health ◽  
Bos Indicus ◽  
Treatment Protocol ◽  
Beef Cows ◽  
High Response Rate ◽  
Control Group ◽  
Embryo Production

This study examined the superovulatory responses of Bos taurus beef cows maintained in a commercial embryo transfer facility. Donors were superovulated 1 to 3 times each with either a traditional 8 injection FSH protocol (controls, n = 126) or 6 injections of FSH with the seventh or eighth FSH treatments replaced by 2 injections of eCG (treatment, n = 134). During the 5-month study, 132 donors were alternatively assigned to a control or treatment group for a single superovulation and an additional 62 animals were superovulated 2 (n = 58) or 3 times (n = 4) in a crossover design. Although 14 beef breeds were represented in the study, 87% of the cows were Angus, Red Angus, Polled Hereford, or Charolais. All donors were synchronized on Day 0 with a CIDR, 5 mg of oestradiol-17β and 100 mg of progesterone. Starting on Day 4 (p.m.), controls were injected twice daily for 4 days with descending doses of porcine FSH (Folltropin-V®, Bioniche Animal Health, Belleville, Ontario, Canada). Cows received 750 μg of cloprostenol (Estrumate®, Intervet Schering-Plough, Summit, NJ, USA) at the seventh FSH injection and the CIDR was removed at the eighth FSH injection. Based on previous experience with specific, individual animals, total FSH dose per donor ranged from 240 to 400 mg. However, 74% of treatments involved 380 mg for controls and 310 mg for treated donors that received eCG. In addition, donors that were superovulated more than once received the same FSH dose in the crossover, treatment-control design. In the treatment group, 200 IU of eCG (Pregnecol™ 6000, Bioniche Animal Health) was substituted for the seventh and eighth FSH injections. Inseminations were conducted on a timed AI basis, with one unit of semen 32 h and a second 48 h following CIDR removal. Results were analysed by ANOVA as shown in Table 1. Although more ova/embryos and unfertilized ova (UFO) were recovered in the control group, the control and treatment groups did not differ in the number of grade 1, 2, or 3 embryos or in the number of degenerate embryos. Previous superovulation studies in South America using eCG to replace the last 2 injections of FSH resulted in more total ova/embryos in Nelore cows but not heifers and in more embryos in Brangus and Sindhi cows. The mean embryo production for the control cows in this study was high and the addition of eCG in the protocol did not improve embryo production. Failure of eCG to increase the number of embryos for Bos taurus cows in this study compared with previous studies may be due to differences with Bos taurus versus Bos indicus breeds or differences in management factors between Canada and South America. The high response rate in the controls may also have contributed to the failure of any advantage of adding eCG to the treatment protocol. Table 1.Mean numbers (± SEM) of ova and embryos recovered from Bos taurus females superovulated with 2 different protocols

402 SUPEROVULATION OF TROPICAL-ADAPTED BOS TAURUS AND BOS INDICUS COWS IN A TROPICAL ENVIRONMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 357
Author(s):  
R. H. Alvarez ◽  
A. C. Martinez ◽  
R. M. L. Pires
Keyword(s):  
Ovarian Function ◽  
Bos Taurus ◽  
Animal Health ◽  
Bos Indicus ◽  
Ovarian Response ◽  
Estradiol Benzoate ◽  
Maximum Temperature ◽  
Embryo Production ◽  
Chi Square ◽  
Average Maximum

Breed differences in ovarian function were found among beef Bos indicus and Bos taurus cows maintained in a subtropical environment (Alvarez P et al. 2000 J. Anim. Sci. 78, 1291-1302). The aim of this study was to compare ovarian response to superovulation and embryo production of tropical-adapted Bos taurus and Bos indicus cows. The experiment was carried out in a tropical wet climate at the experimental station of Instituto de Zootecnia (latitude 22°46′S, longitude 47°17′W) from November to February (average maximum temperature = 30.0 ± 0.8°C and average absolute precipitation = 153.1 ± 78.8 mm3). Forty Caracu (a local Bos taurus breed) and 50 Nelore (Bos indicus breed) lactating cows were treated with an intravaginal device containing progesterone (1.38 mg; CIDR-B®, Pfizer Animal Health, Montreal, Québec, Canada) and 2.5 mg i.m. of estradiol benzoate (Estrogin®, Farmavet, São Paulo, Brazil). Four days later, the animals were superovulated with multiple i.m. injections of 400 IU of FSH (Pluset®, Calier, Spain) in decreasing doses (75-75, 75-50, 50-25, and 25-25 IU) at 12-h intervals over 4 days. The CIDR-B® device was removed 3 days after the first superovulatory injection and cows received i.m. 150 μg of cloprostenol (Veteglan®, Calier, Spain). Cows were inseminated 48 and 62 h after the cloprostenol injection and embryos were recovered nonsurgically 7 days after insemination. Differences in the number of CL (assessed by ultrasound scanning), total number of ova/embryos, and number of transferable embryos were analyzed by ANOVA. Differences in the number of animals with low response (<3 CL) to superovulation were analyzed by chi-square test. All donors (with the exception of 1 Caracu and 2 Nelore) with ovarian response >3 CL showed estrus at insemination. Three (8.9%) Caracu and 5 (10.0%) Nelore cows had <3 CL following the superovulation treatment (P = 0.68). There was no difference (P > 0.05) in the mean (± SEM) CL counts of Caracu (11.4 ± 3.3) and Nelore (12.0 ± 4.1) cows. Similarly, there were no differences (P > 0.05) between Caracu and Nelore cows for total number of ova/embryos collected (8.6 ± 2.6 v. 9.0 ± 4.3) or transferable embryos (6.0 ± 2.4 v. 5.1 ± 2.9). In conclusion, the superovulation of Caracu and Nelore cows carried out in a tropical climate resulted in similar ovarian responses and embryo production. Supported by FAPESP.

213 THE EFFECT OF eCG TREATMENT IN AN ESTRUS SYNCHRONIZATION PROTOCOL ON PREGNANCY RATES AFTER EMBRYO TRANSFER IN HOLSTEIN HEIFERS

2007 ◽  
Vol 19 (1) ◽  
pp. 223
Author(s):  
T. Okazaki ◽  
E. Sasaki ◽  
K. Hasegawa ◽  
T. Takani ◽  
S. Abe
Keyword(s):  
Embryo Transfer ◽  
Prostaglandin F2α ◽  
Estradiol Benzoate ◽  
Pregnancy Rates ◽  
Control Group ◽  
Corpora Lutea ◽  
Estrus Synchronization ◽  
Chi Square ◽  
Synchronization Protocol

Recent studies have shown that the presence of accessory or multiple corpora lutea (CL) and increased progesterone (P4) concentrations reduced early embryonic mortality in cattle. The objective of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) treatment on the number of CL, the P4 concentrations, and pregnancy rates after embryo transfer (ET). Holstein heifers (n = 120) from 7 dairy farms received an intravaginal progesterone-releasing device (CIDR; InterAg, Hamilton, New Zealand) and 2 mg IM of estradiol benzoate (EB; Gynandol®; Sankyo, Tokyo, Japan) at random stages of the estrous cycle. After 7 to 9 days, CIDRs were removed and 15 mg of prostaglandin F2α (PG; Pronalgon®; Pfizer Japan, Nagoya, Japan) were administered, followed by 100 µg IM GnRH (Conceral®; Takeda Pharmaceutical Co., Ltd., Osaka, Japan) 2 days later (Day 0). The heifers were placed at random into 3 groups for eCG treatment. The eCG was not administered in a control group (n = 53); heifers in other 2 groups received 1000 IU eCG (Peamex®; Sankyo, Japan) IM at the time (0 h group, n = 37) or 48 h before (48 h group, n = 30) PG injection/CIDR removal. On Day 7, heifers were examined by ultrasonography (Aloka SSD500; Aloka, Tokyo, Japan) for number of CL; heifers with at least one functional CL received an in vivo-derived frozen–thawed embryo by direct transfer. At the same time, a blood sample was collected to determine P4 concentration. Pregnancy rates were determined on Days 30 and 60 by ultrasonography and rectal palpation, respectively. The data were analyzed by ANOVA and means were compared with Fisher's PLSD. Proportional data were analyzed by the chi-square test. P4 concentrations (mean ± SD) on Day 7 were 1.8 ± 1.0, 5.6 ± 3.3, and 2.2 ± 1.1 ng mL−1 for the control, 48 h, and 0 h groups, respectively (48 h vs. control and 0 h; P &lt; 0.001). The number of CL on Day 7 were 1.1 ± 0.4, 2.5 ± 1.4, and 1.8 ± 0.9 for the control, 48 h, and 0 h groups, respectively (control vs. 48 h and 0 h, and 48 h vs. 0 h; P &lt; 0.01). Pregnancy rates did not differ between 0 and 48 h groups but both were higher than in the control group (Table 1). Results suggest that the estrus synchronization protocol with administration of eCG at the time of CIDR removal or 48 h earlier significantly increased the number of CL and the P4 concentration, and improved pregnancy rates in Holstein heifers after ET. Table 1.Pregnancy rates of Holstein heifers synchronized with CIDR and PG and treated with eCG

scholarly journals Effects of Extra-Long-Acting Recombinant Bovine FSH (bscrFSH) on Cattle Superovulation

Animals ◽  
2022 ◽  
Vol 12 (2) ◽  
pp. 153
Author(s):  
Miguel A. Gutiérrez-Reinoso ◽  
Constanza J. Aguilera ◽  
Felipe Navarrete ◽  
Joel Cabezas ◽  
Fidel O. Castro ◽  
...  
Keyword(s):  
Embryo Quality ◽  
Ovarian Response ◽  
Superior Performance ◽  
Limiting Factors ◽  
Corpora Lutea ◽  
Sample Collection ◽  
Embryo Production ◽  
Expression Ratio ◽  
Long Acting

Over the last few years, several commercial FSH products have been developed for cattle superovulation (SOV) purposes in Multiple Ovulation and Embryo Transfer (MOET) programs. The SOV response is highly variable among individuals and remains one of the main limiting factors in obtaining a profitable number of transferable embryos. In this study, follicle stimulating hormone (FSH) from different origins was included in two SOV protocols, (a) FSH from purified pig pituitary extract (NIH-FSH-p; two doses/day, 12 h apart, four consecutive days); and (b) extra-long-acting bovine recombinant FSH (bscrFSH; a single dose/day, four consecutive days), to test the effects of bscrFSH on the ovarian response, hormone profile levels, in vivo embryo production and the pluripotency gene expression of the obtained embryos. A total of 68 healthy primiparous red Angus cows (Bos taurus) were randomly distributed into two experimental groups (n = 34 each). Blood sample collection for progesterone (P4) and cortisol (C) level determination was performed together with ultrasonographic assessment for ovarian size, follicles (FL) and corpora lutea (CL) quantification in each SOV protocol (Day 0, 4, 8, and 15). Moreover, FSH profiles were monitorised throughout both protocols (Day 0, 4, 5, 6, 7, 8, 9, 10, and 15). In vivo embryo quantity and quality (total structures, morulae, blastocysts, viable, degenerated and blocked embryos) were recorded in each SOV protocol. Finally, embryo quality in both protocols was assessed by the analysis of the expression level of crucial genes for early embryo development (OCT4, IFNt, CDX2, BCL2, and BAX). P4 and cortisol concentration peaks in both SOV protocols were obtained on Day 15 and Day 8, respectively, which were statistically different compared to the other time-points (p < 0.05). Ovarian dimensions increased from Day 0 to Day 15 irrespective of the SOV protocol considered (p < 0.05). Significant changes in CL number were observed over time till Day 15 irrespective of the SOV protocol applied (p < 0.05), being non- significantly different between SOV protocols within each time-point (p > 0.05). The number of CL was higher on Day 15 in the bscrFSH group compared to the NIH-FSH-p group (p < 0.05). The number of embryonic structures recovered was higher in the bscrFSH group (p = 0.025), probably as a result of a tendency towards a greater number of follicles developed compared to the NIH-FSH-p group. IFNt and BAX were overexpressed in embryos from the bscrFSH group (p < 0.05), with a fold change of 16 and 1.3, respectively. However, no statistical differences were detected regarding the OCT4, CDX2, BCL2, and BCL2/BAX expression ratio (p > 0.05). In conclusion, including bscrFSH in SOV protocols could be an important alternative by reducing the number of applications and offering an improved ovarian response together with better embryo quality and superior performance in embryo production compared to NIH-FSH-p SOV protocols.

Embryo production in middle-aged and mature Bos taurus × Bos indicus cows induced to multiple ovulation in a tropical environment

2019 ◽  
Vol 51 (8) ◽  
pp. 2641-2644
Author(s):  
Fernando Naranjo-Chacón ◽  
Felipe Montiel-Palacios ◽  
Rodolfo Canseco-Sedano ◽  
Concepción Ahuja-Aguirre
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
Tropical Environment ◽  
Middle Aged ◽  
Embryo Production ◽  
Multiple Ovulation

287 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND ABSENCE OF CORPUS LUTEUM ON COC RECOVERY IN Gyr (BOS TAURUS INDICUS) COWS

2010 ◽  
Vol 22 (1) ◽  
pp. 300
Author(s):  
T. Miyauchi ◽  
C. A. C. Fernandes ◽  
E. R. Oliveira ◽  
B. F. L. Alves ◽  
J. H. M. Viana
Keyword(s):  
Bos Taurus ◽  
Animal Health ◽  
Cumulus Cell ◽  
Estradiol Benzoate ◽  
Corpora Lutea ◽  
Portable Ultrasound ◽  
Follicular Wave ◽  
Main Technique ◽  
Oocyte Donors

Transvaginal guided follicle aspiration (TGFA) is the main technique used in Brazil to recover oocytes for in vitro embryo production (IVP) in bovine. Different protocols have been proposed to synchronize follicular emergence in oocyte donors, but most of them were developed for use in European breeds of cattle, which show many differences in ovarian physiology when compared with Zebu breeds. The aim of this study was to compare different protocols for preparation of Gyr (dairy zebu breed) oocyte donors. The TGFA were performed in a donor management facility located in Minas Gerais State, southeast Brazil. Pluriparous cycling Gir cows (n = 42) were used as donors. All cows underwent 3 treatments: G1 (control), no treatment before TGFA; G2, 2 mg of estradiol benzoate (EB) for follicular wave synchronization given i.m. 5 days before TGFA; and G3, norgestomet auricular implants given 9 days and 2 mg of EB plus 0.53 mg of cloprostenol given 5 days before TGFA. The interval between TGFA in the same donor was greater than 30 days. All procedures were made by the same technician, using a portable ultrasound device, disposable 19G or 20G needles, and a vacuum pressure of 80 mm Hg. The aspirated follicular fluid was collected in 50-mL Falcon tubes and sent to the laboratory for COC identification and classification under 50 × magnification. Recovered oocytes were classified according to cumulus cell layers and cytoplasm morphology. The total number of oocytes and viable COC recovered and the procedure length (min), including time spent for TGFA and laboratory manipulation, were compared. Data were evaluated by ANOVA, and means compared by Tukey’s test. A total of 126 TGFA sessions were performed, with recovery of 2,809 oocytes (20.31 ± 12.32 of COC and 14.83 ± 7.97 of viable COC per cow/session; mean ± SD). The total number of recovered oocytes and viable COC recovered were lower in G1 compared with G2 and G3 (15.18 ± 11.07 v. 21.18 ± 9.71 and 24.68 ± 9.03; and 9.53 ± 7.22 v. 16.97 ± 6.47 and 18.84 ± 8.90, respectively; P < 0.05) There was no difference (P > 0.05) between G2 and G3 on the number of oocytes or viable COCs recovered. The procedure length, however, was longer in G1 and G2 compared with G3 (49.6 ± 15.1 and 46.9 ± 13.4 v. 35.8 ± 13.1 min, respectively; P < 0.05) The shorter procedure length in G3 was probably associated with the reduced number of cows showing no corpora lutea (38/42, 90%), which resulted in reduction of bleeding and clot formation in the aspirated fluid. These results show that (1) previous follicular wave synchronization by EB can improve the number and quality of recovered oocytes; (2) the absence of corpora lutea does not increase the number of recovered COC and viable COC, but reduces the time spent in the procedure. Intervet Schering Plough Animal Health.

335 CYTOPLASMIC MICROINJECTION OF EXOGENOUS DNA IN IN VITRO AND IN VIVO DERIVED SHEEP EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 263
Author(s):  
F. Pereyra-Bonnet ◽  
A. Gibbons ◽  
M. Cueto ◽  
R. Bevacqua ◽  
L. Escobar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetically Modified ◽  
Fold Increase ◽  
Egfp Expression ◽  
Control Group ◽  
Corpora Lutea ◽  
Exogenous Dna ◽  
Frozen Semen

Microinjection of DNA into the male pronucleus is a commonly used method to generate transgenic animals. However, it is only moderately efficient in several species because it requires proper male pronuclear visualisation, which occurs only in a narrow window of time in mice. The cytoplasmic microinjection of exogenous DNA (eDNA) is an alternative method that has not been fully investigated. Our objective was to evaluate if cytoplasmic microinjection of eDNA is capable of producing genetically modified embryos. In vitro and in vivo derived sheep embryos were cytoplasmically microinjected with pCX-EGFP previously incubated (5 min in a PVP droplet) with oolemma-cytoplasm fragments obtained from donor oocytes by microsurgery. A control group using microinjected plasmid alone was included in the in vivo procedure. For in vitro microinjection, IVF embryos were microinjected with circular plasmid with promoter (50 or 500 ng μL–1) or without promoter (50 ng μL–1) at 6 h after fertilization. The IVF was performed following (Brackett and Olliphant 1975 Biol. Reprod. 12, 260–274) with 15 × 106 spermatozoa mL–1, and presumptive zygotes were cultured in SOF. The expression of enhance green fluorescent protein (EGFP) was determined under blue light. For in vivo microinjection, embryos from superovulated sheep (by standard procedures) were recovered and microinjected with 50 ng μL–1 of linearized plasmid without promoter at 12 h after laparoscopic insemination with frozen semen (100 × 106 spermatozoa per sheep). Plasmid without promoter was used to avoid any possible cytotoxic effect produced by EGFP expression. The microinjection of IVF embryos with 50 ng μL–1 of plasmid was the best condition to produce embryos expressing eDNA (n = 96; 46.9% cleaved; 12.2% blastocysts; 53.0 and 4.1% of green embryos and blastocysts, respectively). Variables between the groups with or without promoter IVF were not statistically different (Fisher test: P < 0.05); however, when 500 ng μL–1 was microinjected, no blastocysts were obtained. In the in vivo embryo production group, 111 presumptive zygotes were microinjected (n = 37; with plasmid alone) from 16 donor sheep (11.5 ± 4.0 corpora lutea; 8.4 ± 4.8 presumptive zygotes recovered; 74.3% recovery rate). The mean time from injection to cleavage was 18.0 ± 4.5 h, and the percentage of cleavage and damage (due to the embryo injection) were >70% and <10%, respectively. Fifty-eight good quality embryos were transferred into the oviducts of 19 surrogate ewes; 12 of them are pregnant (63.1%). The presence of green IVF embryos demonstrates that eDNA was transported to the nucleus after cytoplasmic injection. We believe that the multi-fold increase (50- to 100-fold) in plasmid concentration compared with that used by others was the key step to our successful cytoplasmic microinjection. Accordingly, the new/old methodology described in this study provides an easy DNA construct delivery system of interest for the implementation of early reprogramming events. In addition, results obtained in the near future using in vivo cytoplasmic microinjection with high concentrations of eDNA could revalidate this technique for producing genetically modified large animals.

121 DIFFERENTIAL mRNA EXPRESSION BETWEEN IN VIVO AND IN VITRO-DERIVED BOS INDICUS AND BOS TAURUS EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 160
Author(s):  
L. Nasser ◽  
P. Stranieri ◽  
A. Gutiérrez-Adán ◽  
M. Clemente ◽  
L. Jorge de Souza ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Repeated Measures ◽  
Bos Taurus ◽  
Receptor Gene ◽  
Expression Patterns ◽  
Bos Indicus ◽  
Growth Factor Receptor ◽  
P53 Gene

Brazil is a leading country in the world of commercial use of in vitro-produced bovine embryos with 200 000 transfers per year. The majority of in vitro-produced embryos are pure breed Nelore and are transferred fresh with 40% pregnancy rate. However, pregnancies are drastically reduced with frozen in vitro embryos. This experiment is part of our effort to learn more about molecular composition and morphology of in vitro-derived embryos that may be responsible for such discrepancy. We examined molecular expression of mRNA transcripts of 6 selected genes; apoptosis Bax,TP53(p53), SHC1SHC(p66), insulin growth factor receptor (IGF2R), stabilization of the plasma membrane PLAC8 and glucose conversion H6PD in in-vivo (control) and in-vitro Nelore and Bos taurus embryos. In vivo embryos were collected from superovulated cows at Day 7. In vitro embryo was produced from oocytes aspirated from live cows. A total of 284 oocytes (4 replicates) were matured and fertilized by standard IVF procedures. Presumptive zygotes were cultured in CR2 medium with 5% BSA in 50 μL drops (25 zygotes per drop) at 39°C under paraffin oil and 5% CO2 in humidified air. Embryos that developed on Days 7 to blastocyst were transferred to recipients, and 10 blastocysts from each replicate were frozen for evaluation of gene expression patterns. Poly(A) mRNA was prepared from 3 groups of pools of 10 in vitro embryos and 10 of control in vivo-derived embryos. The quantification of all gene transcripts was carried out by real-time quantitative RT-PCR using the comparative CT method. Data on mRNA expression were normalized to the endogenous H2a.z and was analyzed by one-way repeated-measures ANOVA. The cleavage rates at Day 2 and number of blastocysts developed at Day 7 were 80.3 ± 3.2 and 42.2 ± 6.4, respectively. The level of expression of IGF2R was significantly (P < 0.05) higher in in vivo-derived embryos than in both groups of in vitro embryos. The expression of all 3 apoptosis genes were lower (P < 0.05) in in vivo than in vitro embryos with exception of p53 gene that was not different between Nelore in vitro and in vivo embryos but was significantly higher (P < 0.05) in Bos taurus in vitro embryos. There was no difference in expression of PLAC8 gene among any tested group of embryos and in expression of H6PD gene between Nelore in vitro and in vivo embryos. We concluded that significant differences in molecular makeup between in vitro and in vivo-derived Nelore embryos exist. Of particular importance seems to be pattern of expression of IGF2R receptor gene known as a good indicator of embryo quality, which promotes proliferation and differentiation. Similarly, higher expression of 2 BAX and p66 genes of apoptosis in in vitro embryos seems to be a further indication of inferior quality of Nelore in vitro-derived embryos that showed to be more profound in Bos taurus in vitro-derived embryos.

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