109 COMPARISON OF METHODS FOR SYNCHRONIZING RECIPIENTS OF IN VITRO PRODUCED EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 185 ◽  
Author(s):  
R. V. Sala ◽  
L. C. Carrenho-Sala ◽  
M. Fosado ◽  
L. C. C. Tosta ◽  
R. D. Tosta ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Prostaglandin F2α ◽  
Gonadotropin Releasing Hormone ◽  
Comparison Of Methods ◽  
Releasing Hormone ◽  
Embryo Stage ◽  
Utilisation Efficiency ◽  
Potential Recipient ◽  
Second Use

The present study compared fertility, as pregnancy per embryo transfer (P/ET), and efficiency of recipient utilisation, as pregnancy per treated potential recipient (P/TX), in heifers receiving in vitro-produced embryos using synchronized oestrus after prostaglandin F2α (OESTRUS) or synchronized ovulation and fixed timed embryo transfer (FTET) with new or reused CIDR. In Expt. 1, heifers (n = 520) were randomly assigned to 1 of 3 groups: OESTRUS, FTET with new CIDR, or FTET with second-use CIDR (previously used for 5 days). Heifers in OESTRUS group (n = 166) were synchronized with two prostaglandin F2α 14 days apart and detection of oestrus performed using tail chalk during 5 days after the second prostaglandin F2α. Heifers in FTET were synchronized with a new CIDR (n = 178) or second-use CIDR (n = 176) using a modified 5-day CIDR-Synch; Day –8: CIDR inserted; Day –3: CIDR removed, prostaglandin F2α; Day –2: second prostaglandin F2α; Day 0: gonadotropin-releasing hormone to induce ovulation. In Expt. 2, heifers (n = 422) were randomly assigned to 1 of 2 groups: FTET with new CIDR or FTET with third-use CIDR (previously used twice for 5 days each time) using the FTET protocol described for Expt. 1. Fresh in vitro-produced embryos were transferred between 6 and 8 days after OESTRUS or gonadotropin-releasing hormone (FTET). All heifers were evaluated by transrectal ultrasonography on Day 32 and 60 for pregnancy detection. Measurements of P/ET and P/TX for both experiments were analysed by logistic regression (LOGISTIC procedure, SAS 9.4) using biologically meaningful covariates such as embryo stage and quality, interval from oestrus or GnRH to transfer, and technician in the statistical analyses. In Expt. 1, two preplanned contrasts were performed to compare differences between OESTRUS v. FTET, and between FTET with new v. second-use CIDR. The P/ET at Day 32 was similar (P = 0.50) with 41.3% (45/109) for OESTRUS and 43.4% (134/309) for FTET groups. Similarly, P/ET on Day 60 was 30.3% (33/109) for OESTRUS and 32.4% (100/309) for FTET groups (P = 0.37). However, P/TX heifer on Day 60 was greater (P = 0.04) in the FTET (28.2%; 100/354) compared to OESTRUS (19.9%; 33/166). This difference is attributed to a greater (P < 0.001) utilisation efficiency (transferred/treated) of heifers in FTET (87.3%) v. OESTRUS (65.6%). In the second contrast, P/ET on Day 32 were similar (P = 0.87) for FTET heifers synchronized with a new CIDR (43.9%, 69/157) v. second-use CIDR (42.8%, 65/152). In addition, P/TX heifer on Day 60 was also similar (P = 0.52) for heifers receiving a new or second-use CIDR (29.8%, 53/178 v. 26.7% 47/176). In Expt. 2, P/ET on Day 32 was similar (P = 0.73) for FTET with a new CIDR (41.0%, 77/188) or third-use CIDR (42.3%, 83/196). The P/TX heifer on Day 60 was also not different (P = 0.58) for new CIDR (28.6%, 61/213) v. third-use CIDR (31.1%, 65/209). Thus, use of FTET with new or used CIDR can produce similar P/ET and greater efficiency of recipient utilisation compared to OESTRUS.

110 TREATMENT WITH GnRH ON DAY 5 REDUCES PREGNANCY LOSS IN HEIFERS RECEIVING IN VITRO-PRODUCED EXPANDED BLASTOCYSTS

2016 ◽  
Vol 28 (2) ◽  
pp. 185 ◽  
Author(s):  
A. Garcia-Guerra ◽  
R. V. Sala ◽  
G. M. Baez ◽  
M. Fosado ◽  
L. F. Melo ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Pregnancy Loss ◽  
Body Condition Score ◽  
Prostaglandin F2α ◽  
Gonadotropin Releasing Hormone ◽  
Crown Rump Length ◽  
Releasing Hormone ◽  
Embryo Size ◽  
Embryo Stage

The hypothesis was that GnRH on Day 5 of a synchronized cycle in embryo transfer recipients would increase progesterone (P4) concentrations, embryo size, and fertility. Holstein and cross-bred Holstein heifers (n = 1562) were synchronized using a modified 5-day CIDR Co-Synch as follows: Day –8 CIDR inserted; Day –3 CIDR removed; prostaglandin F2α treatment; Day –2 second prostaglandin F2α; Day 0 gonadotropin-releasing hormone (G1, 100 μg of gonadorelin acetate) to induce ovulation. On Day 5.5, heifers were assigned in a completely randomised design to 1 of 2 treatments: Control (untreated) or GnRH (200 μg of gonadorelin acetate). Transfer of fresh in vitro-produced embryos was performed between d 6 and 8 after G1. Data collected from each heifer included embryo stage and quality, body condition score, technician, interval from G1 to transfer, and number of previous transfers. All heifers were evaluated by transrectal ultrasonography on Day 5, 33, and 62 and a subset of heifers was scanned on Day 12 (n = 718; to determine ovulation to treatment) and another subset on Day 33 (n = 296; 16-s video to determine embryo and amniotic vesicle size). Serum P4 was determined from a subset of heifers on Day 12 (n = 467). Fertility data were analysed by logistic regression (LOGISTIC procedure, SAS 9.4), whereas continuous outcomes were analysed by ANOVA (MIXED procedure). Ovulation to Day 5.5 gonadotropin-releasing hormone was 83.9% (302/360) in GnRH-treated heifers v. 3.3% (12/358) in Control (P < 0.001). Progesterone on Day 12 was greater in GnRH-treated heifers 7.2 ± 0.1 ng mL–1 v. Controls 6.0 ± 0.1 ng mL–1 (P < 0.001). There was an effect of embryo stage at Day 33 and 60 of pregnancy, with Stage 7 having greater P/ET than Stage 6 embryos. Treatment with GnRH did not alter pregnancy per embryo transfer with either embryo stage but decreased pregnancy loss in Stage 7 embryos, as shown in Table 1. Embryo size measured as crown-rump length (CRL) did not differ, as shown in Table 1. Similarly, amniotic vesicle volume (AVV) was not different between GnRH (549.1 ± 16 mm3) and Control (543.5 ± 14 mm3; P = 0.86), nor was there an interaction between treatment and embryo stage (P = 0.71). In addition, neither AVV (P = 0.22) nor CRL (P = 0.41) were associated with pregnancy loss between Day 33 and 60. In conclusion, treatment with GnRH on Day 5 resulted in increased P4 and a reduction in pregnancy loss in heifers receiving a Stage 7 embryo without changing conceptus size. Table 1.Pregnancies per embryo transfer (P/ET), crown-rump length (CRL), and pregnancy loss in embryo recipients receiving gonadotropin-releasing hormone (GnRH) on Day 5.5 v. control

104 FACTORS AFFECTING PREGNANCY RATES AND EMBRYO/FETAL LOSSES IN RECIPIENTS RECEIVING IN VITRO-PRODUCED EMBRYOS BY FIXED-TIME EMBRYO TRANSFER

2017 ◽  
Vol 29 (1) ◽  
pp. 160
Author(s):  
A. Tribulo ◽  
A. Cedeño ◽  
B. Bernal ◽  
S. Andrada ◽  
J. L. Barajas ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Body Condition Score ◽  
Prostaglandin F2α ◽  
Fixed Time ◽  
Gonadotropin Releasing Hormone ◽  
Pregnancy Rates ◽  
Device Removal ◽  
Releasing Hormone ◽  
Condition Score

A retrospective analysis evaluated pregnancy rates and embryo losses with in vitro-produced embryos in a commercial embryo transfer program on 15 different beef farms. Recipients were beef cows and heifers (n = 1841) that were synchronized with 5 different protocols and transferred at a fixed-time (FTET). Recipients were examined by ultrasonography on Day 0, and those with a corpus luteum (CL) or a follicle ≥8 mm in diameter and with body condition score 2 to 4 (1 to 5 scale) were synchronized. The synchronization treatments were as follows. (T1) Recipients received an intravaginal device with 0.5 g of progesterone plus 2 mg of oestradiol benzoate on Day 0; device removal, plus 500 μg of cloprostenol (prostaglandin F2α), 400 IU of eCG, and 0.5 mg of oestradiol cypionate on Day 8; and FTET on Day 17. (T2) This treatment was similar to T1 but 1 mg of oestradiol cypionate was injected at device removal instead of 0.5 mg of oestradiol cypionate. (T3) This treatment was similar to T1 except that animals were tail-painted on Day 8 and observed on Day 10. Those with the tail-paint intact on Day 10 received 100 μg of gonadorelin (gonadotropin-releasing hormone) and all recipients were FTET on Day 17. (T4) Recipients received a progesterone device on Day 0; device removal, prostaglandin F2α, and eCG on Day 5; gonadotropin-releasing hormone on Day 8; and FTET on Day 15. (T5) Recipients received a progesterone device and 2 mg of oestradiol benzoate on Day 0; device removal, prostaglandin F2α, and eCG on Day 6; gonadotropin-releasing hormone on Day 9; and FTET on Day 16. On the day of FTET all recipients with CL ≥18 mm in diameter (G1), ≥16 and <18 mm in diameter (G2), and ≥14 mm and <16 mm in diameter (G3) received in vitro-produced fresh embryos. Pregnancy was diagnosed by ultrasonography at 30 and 60 days of gestation, and data were analysed by logistic regression. The overall proportion of recipients synchronized that were FTET was 80.8% (1487/1841), with a 30-day pregnancy rate to FTET (P/FTET) of 45.6% (678/1487) and the rate of 30- to 60-day embryo/fetal loses on the 528 recipients that were re-checked at 60 days was 12.8% (68/528). There were no significant differences in P/FTET among operators, animal category, time of the year, embryo stage, or body condition score; however, there was a significant effect of farm (P < 0.001) and CL diameter (P < 0.05), but no interaction between CL diameter and farm or treatment (P > 0.1). Recipients with G1 (443/953, 46%) and G2 (221/462, 47%) CL had higher pregnancy rates than those with G3 CL (23/71, 32%). There was a significant effect of synchronization treatment on the proportion of recipients transferred and on P/FTET (P < 0.01) that was highly influenced by farm (farm by treatment interaction P < 0.01). The proportions of recipients selected for embryo transfer were as follows: T1: 386/486, 79.4%; T2: 233/331, 70.3%; T3: 342/377, 90.7%; T4: 126/160, 78.7%; and T5: 400/487, 82.1%. The P/FTET were as follows: T1: 190/386, 49.2%; T2: 96/233, 41.2%; T3: 175/342, 51.1%; T4: 49/126, 38.8%; and T5: 168/400, 42.0%. Although 30- to 60-day embryo/fetal losses were not influenced by synchronization treatments, they were highly influenced by farm (P < 0.001) and ranged from 0 to 34.5%. In conclusion, P/FTET in a commercial program with beef in vitro-produced embryos was influenced by factors related to the recipient (CL diameter) and the environment (farm), whereas embryo/fetal losses were influenced by farm but not treatment or recipient factors.

157 PREGNANCIES AND CALVES AFTER TRANSFER OF IN VITRO-PRODUCED RIVER BUFFALO EMBRYOS AFTER CRYOPRESERVATION

2012 ◽  
Vol 24 (1) ◽  
pp. 190 ◽  
Author(s):  
C. Galli ◽  
R. Duchi ◽  
G. Lazzari ◽  
I. Lagutina ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Prostaglandin F2α ◽  
Gonadotropin Releasing Hormone ◽  
North Coast ◽  
Transfer Program ◽  
Embryo Production ◽  
Releasing Hormone ◽  
In Vitro Embryo Production

In the buffalo, the use of embryo-based biotechnologies for breeding and genetic improvement is still very limited because multiple-ovulation embryo transfer delivers poor results compared with cattle and in vitro embryo production has been used mainly for research purposes. At present, very few reports are available on the transfer of in vitro-produced (IVP) and cryopreserved buffalo embryos. Therefore, the scope of this work was to perform a pilot study to evaluate the viability of frozen-thawed IVP embryos by nonsurgical embryo transfer to recipients in an IVF-embryo transfer program on a farm located on the north coast of Colombia, South America. Buffalo oocytes were recovered at the slaughterhouse from selected donors, matured in vitro for 18 to 20 h in TCM-199 + 10% FCS and 0.5 IU of FSH and 0.5 IU of LH in 5% CO2 at 38.5°C. Four different bulls were used for IVF. After thawing, the semen was separated on a Percoll® gradient and then diluted into SOF-IVF media supplemented with 1 μg mL–1 of heparin and phenylalanine. Presumptive zygotes were cultured in modified SOF supplemented with MEM amino acids for 6 days. Half of the medium was replaced on Day 4 and 6. Developing embryos were selected for freezing on Day 6 and 7. Grade 1 embryos were frozen at the blastocyst stage by slow cooling in 10% glycerol or 1.5 M ethylene glycol. Recipients (heifers n = 79 and uniparous cows n = 17) were synchronized using the CIDR-Synch protocol: on Day 0, gonadotropin-releasing hormone was injected and a CIDR was inserted; on Day 7, prostaglandin F2α was administered; on Day 9, the CIDR was removed; on Day 11, a second injection of gonadotropin-releasing hormone was given; and on Day 17, the embryo was transferred. Each female received, nonsurgically, 1 or 2 embryos in the ipsilateral horn to the functional corpus luteum evaluated by ultrasonography. Pregnancies were evaluated by ultrasonography 30 days after transfer and confirmed by rectal palpation 30 days later. This work was performed in 2 successive experiments during the breeding seasons (January and December, respectively). Overall, 96 recipients were transferred, with 136 embryos obtaining 23 pregnancies (24.2%). There were no statistical differences in pregnancy rate between heifers and cows (25.3 vs 17.7%) and between single (n = 56) and double (n = 39) embryo transfers (21.4 vs 27.5%) by chi square test (P > 0.05). To date, 4 females and 5 males have been born by spontaneous calving (1 stillborn male due to dystocia), 3 pregnancies have been aborted (13%) and 11 pregnancies are ongoing (>7 months). The pregnancy rate obtained in this study in farm conditions (24.2%) is lower than generally obtained with frozen IVP cattle embryos, but it is still a good result in buffalo, where even conventional AI provides a lower success rate as compared with cattle. Finally, this work demonstrates that in vitro embryo production can be successfully implemented in buffalo breeding programs for the exploitation of superior genetics. This work was supported by Regione Lombardia, Por Fers 2007–2013, n°13827741, InnovaB.

107 FACTORS THAT INFLUENCE FERTILITY IN AN IVF EMBRYO TRANSFER PROGRAM IN DAIRY HEIFERS

2016 ◽  
Vol 28 (2) ◽  
pp. 183 ◽  
Author(s):  
L. C. Carrenho-Sala ◽  
R. V. Sala ◽  
M. Fosado ◽  
D. C. Pereira ◽  
S. Garcia ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Pregnancy Loss ◽  
Embryo Quality ◽  
Prostaglandin F2α ◽  
Transfer Program ◽  
Dairy Heifers ◽  
Embryo Transfer Program ◽  
Embryo Stage ◽  
Major Factors

A retrospective study was performed to evaluate factors that influence pregnancy per embryo transfer (P/ET) in an IVF-embryo transfer program. A total of 5026 fresh in vitro-produced embryos were transferred during 2014 and evaluated for effects of embryo quality, embryo stage, size of corpus luteum (CL; 18–19.9 mm or ≥20 mm), interval from GnRH to embryo transfer, number of previous embryo transfer (0, 1, 2, 3, ≥4); and interaction of embryo stage and interval from GnRH to embryo transfer. One group (n = 850) had detection of oestrus after prostaglandin F2α application but most heifers (n = 4176) received fixed timed embryo transfer after a 5-day CIDR-Synch protocol: Day –8 CIDR inserted; Day –3 CIDR removed and prostaglandin F2α; Day –2 prostaglandin F2α; Day 0 GnRH. Ultrasound was performed on Day 6 after GnRH or oestrus to measure CL size and on Day 32 and 60 to determine pregnancy. Data for P/ET were analysed by logistic regression (LOGISTIC procedure, SAS 9.4). Embryo quality influenced P/ET at Day 32 [Grade 1 48.4% (1273/2631) v. Grade 2 37.6% (900/2395); P < 0.01] and at Day 60 [Grade 1 38.9% (1023/2631) v. Grade 2 29.0% (694/2395); P < 0.01], and altered pregnancy loss [Grade 1 19.6% (250/1273) v. Grade 2 22.9% (206/900); P = 0.03]. Stage of the embryo also had an effect on P/ET at Day 32 [Stage 6 35.5%a (582/1641), Stage 7 46.3%b (1431/3092), and Stage 8 54.6%c (160/293); P < 0.01] and at Day 60 [Stage 6 28.2%a (462/1641), Stage 7 36.6%b (1131/3092), and Stage 8 41.6%b (122/293); P < 0.01], but did not affect pregnancy loss (P = 0.22). Interestingly, interval from GnRH (or oestrus) until embryo transfer did not affect P/ET at Day 32 (P = 0.10), 60 (P = 0.23), or pregnancy loss (P = 0.3), nor was there an interaction between interval and embryo stage at Day 32 (P = 0.77), 60 (P = 0.96) or pregnancy loss (P = 0.55). As shown in Table 1, embryo stage 6 was always the lowest and stage 8 always the greatest P/ET regardless of interval from GnRH to embryo transfer. Size of CL also did not affect P/ET at Day 32 (P = 0.09), 60 (P = 0.21), or pregnancy loss (P = 0.90). Number of previous embryo transfer also did not alter P/ET at Day 32 [0 = 43.3% (886/2046), 1 = 44.1% (639/1450), 2 = 43.4% (444/1024), 3 = 42.6% (146/343), and ≥4 = 35.6% (58/163); P = 0.33] or 60 (P = 0.51) or pregnancy loss (P = 0.12). In conclusion, embryo stage and quality are the major factors that impacted P/ET in this study, with surprisingly little effect of interval from GnRH to embryo transfer, size of the CL, and number of previous embryo transfer. Thus, recipient programs for IVF-embryo transfer can be designed with substantial flexibility. Table 1.Effect of embryo stage and recipient synchrony on pregnancies per embryo transfer on Day 32 in recipient dairy heifers

252 EFFECT OF SUPERSTIMULATORY TREATMENT TO DONOR COWS IN OXYGEN CONSUMPTION OF MATURED OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 273
Author(s):  
K. Imai ◽  
S. Sugimura ◽  
M. Ohtake ◽  
Y. Aikawa ◽  
Y. Inaba ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Prostaglandin F2α ◽  
Gonadotropin Releasing Hormone ◽  
Oestrous Cycle ◽  
Control Group ◽  
Releasing Hormone ◽  
And Control ◽  
Bovine Oocytes

We previously reported that follicular wave synchronization and follicular growth treatment (FGT) before ovum pick-up (OPU) were effective in improving oocyte competence, which was associated with an increase in related embryos obtained by somatic cell nuclear transfer (Sugimura et al. 2012 Cell. Reprogram. 14, 29–37). However, oxygen consumption in oocytes remained unknown. The present study was designed to examine the differences in oxygen consumption between bovine oocytes obtained by OPU with or without FGT after in vitro maturation. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two OPU sessions were conducted in each cow to collect immature oocytes, as described by Sugimura et al. (2012). The first OPU session (OPU group) was performed in cows on arbitrary days of the oestrous cycle, using a 7.5-MHz linear transducer with the needle connected to an ultrasound scanner. Follicles larger than 8 mm in diameter were then aspirated and a controlled internal drug release device (CIDR) was inserted on Day 5 (the day of the first OPU session = Day 0). Then 30 Armour units (AU) of FSH (Antrin, Kyoritsu Seiyaku, Tokyo, Japan) was administrated to cows twice a day from Day 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 AU day–1). Cloprostenol (prostaglandin F2α; 0.75 mg) was administered in the morning of Day 9. The second OPU session (FGT-OPU group) was performed 48 h after prostaglandin F2α administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected cumulus–oocyte complexes in the OPU and FGT-OPU groups were matured in vitro as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. To collect in vivo-matured oocytes (control group), the CIDR was inserted into the cows on arbitrary days of the oestrous cycle (= Day 0), and oestradiol benzoate (0.8 mg) was administered on Day 1. The cows received the FGT treatment (as described above) from Day 6 to 10; however, the CIDR was removed in the evening of Day 8. Buserelin (gonadotropin-releasing hormone; 200 µg) was then administrated in the morning of Day 10, and OPU was performed at 24 h after gonadotropin-releasing hormone administration (Day 11). Oxygen consumption of matured oocytes was measured noninvasively with a scanning electron microscopy system (HV-405SP; Hokuto Denko Co., Tokyo, Japan). Data were analysed by ANOVA followed by a Tukey-Kramer test. There was no difference in the mean oxygen consumption between the FGT-OPU group (0.34 ± 0.02 × 10–14 mol–1, mean ± SEM) and control group (0.40 ± 0.01 × 10–14 mol–1). However, oxygen consumption in the FGT-OPU and control groups was significantly lower (P < 0.01) than that in the OPU group (0.50 ± 0.02 × 10–14 mol–1). These results revealed significantly lower oxygen consumption in OPU-derived in vitro-matured bovine oocytes after FGT treatment compared with those obtained without FGT treatment. Oxygen consumption of oocytes obtained from FGT-OPU was similar to that of in vivo-matured oocytes, which may reflect their cytoplasmic maturation status with high developmental competence.

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