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The pregnancy rates obtained after the transfer of cryopreserved in vitro-produced (IVP) embryos are usually low and/or inconsistent. The objective of this study was to evaluate the pregnancy rates of Holstein, Gyr and Holstein × Gyr cattle after the transfer of vitrified IVP embryos produced with X-sorted sperm. Seventy-two Gyr and 703 Holstein females were subjected to ovum pickup (OPU) sessions, followed by in vitro embryo production using semen from sires of the same breeds. Embryos (1636 Holstein, 241 Gyr and 1515 Holstein × Gyr) were exposed to forskolin for 48 h prior to vitrification. The pregnancy rate achieved with Gyr dam and sire was 46.1%, which was similar (p = 0.11) to that of Holstein dam and Gyr sire (40.3%). Crossing Gyr dams with Holstein sires resulted in a pregnancy rate of 38.9% and did not differ (p = 0.58) from the pregnancy rate obtained with the cross between Holstein dams and Gyr sires. The rate obtained with Holstein dam and sire was 32.5%. The average pregnancy rate was 36.6%, and no difference was found in the proportion of female foetuses (88.8%, in average) among breeds (p > 0.05). In conclusion, transfer of cryopreserved X-sorted embryos represents an interesting choice for dairy cattle. Despite the small differences between pregnancy rates, we highlight the efficiency of this strategy for all of the racial groups studied., (© 2015 Blackwell Verlag GmbH.)

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In vitro-produced (IVP) bovine embryos are more sensitive to cryopreservation than their in vivo counterparts due to their higher lipid concentrations, whereas Bos indicus IVP embryos are even more sensitive than Bos taurus IVP embryos. To examine the effects of a lipolytic agent, before vitrification of Bos indicus IVP embryos, on embryo survival, viability, and pregnancy rates, two experiments were conducted. In experiment 1, Bos indicus (Nelore) embryos were produced from abattoir-derived ovaries and allocated into two groups. In the treatment group, 10 μM of forskolin was added to the in vitro culture medium on Day 5 and incubated for 48 hours. On Day 7 of culture, IVP-expanded blastocysts from both the control (n = 101) and treatment (n = 112) groups were vitrified with ethylene glycol and DMSO via the Cryotop procedure. Although there was no significant difference between the rates of blastocoel reexpansion and hatching of the embryos exposed to forskolin (87.5% and 70.5%, respectively) compared with the control embryos (79.2% and 63.3%, respectively), the numerically superior rates of the embryos exposed to forskolin led to another experiment. In experiment 2, blastocysts produced from the ovum pick up were exposed or not exposed to the lipolytic agent and vitrified as in experiment 1. Embryos treated with forskolin had higher pregnancy rates than the control group (48.8% vs. 18.5%). In view of these results, 1908 Bos indicus embryos were produced from ovum pick up, exposed to the lipolytic agent, and blastocysts were transferred to recipients, and the pregnancy rates of the embryos of various breeds were compared. The mean pregnancy rate obtained was 43.2%. All data were analyzed by chi-square or by binary logistic regression (P ≤ 0.05). In conclusion, treatment with forskolin before vitrification improved cryotolerance of Bos indicus IVP embryos, resulting in good post-transfer pregnancy rates., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The objective was to clarify in vitro production of bovine embryos in Brazil. Data from 656 ovum pick-up/in vitro production (OPU/IVP) procedures, performed on 317 Nelore (Bos indicus) donors, without hormone stimulation or control of ovarian follicular waves, were analysed. Donors were subjected to OPU from one to nine times (no specific schedule), with < 15 d between consecutive procedures. There were 20,848 oocytes, of which 15,747 (75.53%) were considered viable, 5,446 embryos were obtained, 5,398 embryos were immediately transferred, resulting in 1,974 pregnancies (36.57%) at Day 30 and 1,788 (33.12%) pregnancies at Day 60. The average number of total and viable oocytes produced per OPU session was (mean ± SEM) 30.84 ± 0.88 and 23.35 ± 0.7 (average of 8.1 ± 0.3 embryos and 3.0 ± 0.1 pregnancies per OPU-IVP procedure). Since oocyte production varied widely among donor, they were designated as very high, high, intermediate, and low, with 58.94 ± 2.04, 32.61 ± 0.50, 22.13 ± 0.50, and 10.26 ± 0.57 oocytes, respectively, produced by 78, 80, 79, and 80 donors. The number of viable oocytes recovered ranged from 0 to 128; since donors with numerous viable oocytes produced many viable embryos and pregnancies, oocyte production was useful for donor selection. However, there was no significant effect of the number of OPU sessions per donor on mean numbers of oocytes produced. In conclusion, we confirmed field reports of high oocyte production by some Nelore donors and demonstrated individual variation in oocyte yield, which was associated with embryo production and pregnancy rates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO(2) in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10(6) sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos., (Copyright © 2010 Elsevier Inc. All rights reserved.)

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n=30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4+/-0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2+/-1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6+/-15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4+/-5.3) was higher (P<0.05) than the MOET method (6.7+/-3.7). However, pregnancy rates were lower (P<0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P>0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.