Lidia cattle are a breed of Bos taurus that has been selected specially to produce bulls with the temperament and aggressiveness necessary to face a bullfighter in a ring. The genetic wealth of this fighting breed is divided into small lineages, traditionally called encastes, which has resulted in the risk of a loss of genetic variability (Ministerio de Medio Ambiente y Medio Rural y Marino, 2011; http://www.toroslidia.com/wp-content/uploads/2012/01/Programa-de-mejora-de-la-Raza-Bovina-de-Lidia.pdf). The technique to produce embryos in vitro may be a useful tool in the conservation of genetic material from this breed in a selection program. The aims of the study were to demonstrate the effectiveness of in vitro production of Lidia cattle embryos, and to evaluate variation in embryo production among males of the breed. Lidia cows, 7 to 13 years of age (n = 12), were used in an ovum pick-up (OPU)-in vitro production (IVP) program in the south of France. Ovarian superstimulation was induced with decreasing doses of pFSH (Stimufol; Reprobiol, Liège, Belgium) twice daily over 3 days (total dose: 350 µg). Transvaginal ultrasound-guided collection of cumulus–oocyte complexes (COC) was done 12 to 24 h after the last FSH injection. The COC were evaluated immediately after OPU and placed into 2.0-mL tubes (Corning Inc., Corning, NY, USA) containing 500 µL of maturation medium. A gas mix (5% CO2 in air) was injected into each tube and the tube was sealed tightly and placed in a portable incubator (Minitub, Tiefenbach, Germany) at 38.0°C for 12 h. On arrival in the Auriva IVP laboratory, tubes were opened and placed into an incubator with 5% CO2 at 38.5°C at maximum humidity to complete a 24-h maturation period. Semen was collected by electro-ejaculation previously from 5 different Lidia bulls (A, B, C, D, and E) and had been frozen by the same technique. The COC were fertilized with the frozen–thawed semen in TALP medium. Presumed zygotes were cultured in SOF medium (Minitub) to Day 7 (Day 0 = fertilization day) at 38.5°C in a 5% CO2, 5% O2, and 90% N2 atmosphere with maximum humidity. A total of 19 OPU/IVP sessions were performed, 5 cows were collected once, and 7 cows collected twice, and 143 COC were processed for in vitro embryo production. Blastocyst and expanded blastocyst numbers were recorded on Day 7. Oocyte recovery and embryo production by bull were analysed by ANOVA and blastocyst yield by Chi-square. The number (mean ± SEM) of oocytes allocated to each bull per IVP session was (P > 0.05): bull A (4.5 ± 1.9), bull B (5.8 ± 2.1), bull C (9.3 ± 2.5), bull D (6.5 ± 2.1), and bull E (7.0 ± 4.4). The cleavage rate differed among bulls (P < 0.05): bull A (4%), B (80%), C (89%), D (81%), and E (76%). The number (mean ± SEM) of blastocysts was lowest (P < 0.05) for bull A and highest (P < 0.05) for bull C (0, 3.7 ± 1.8, 7.0 ± 1.0, 4.3 ± 1.3, 4.7 ± 2.3 for bulls A to E, respectively). The blastocyst development rate (number of blastocysts/number of oocytes entering the IVF process) was also different among bulls (0, 63, 75, 65, and 67%, respectively; P < 0.05). Although there was a male effect on blastocyst production, our data demonstrate that successful in vitro embryo production in Lidia cattle is possible and suggests that this tool would be useful in a genetic program for the multiplication and the conservation of this breed.
In Vitro Production of Embryos from Prepubertal Holstein Cattle and Mediterranean Water Buffalo: Problems, Progress and Potential
