Different doses of equine chorionic gonadotropin on ovarian follicular growth and pregnancy rate of suckled Bos taurus beef cows subjected to timed artificial insemination protocol.

This study evaluated the effect of different doses of eCG (control, 300 or 400 IU) administered at progesterone (P4) device removal in suckled Bos taurus beef cows undergoing a timed artificial insemination (TAI) protocol. A total of 966 cows received a P4 insert and 2.0 mg intramuscular estradiol benzoate at the onset of the synchronization. After 9 days, P4 insert was removed, and 12.5 mg of dinoprost tromethamine and 1 mg of estradiol cypionate were administered, followed by TAI 48 hours later. Then, the cows received one of three treatments as follows: control (n = 323), 300 (n = 326), or 400 IU of eCG (n = 317). A subset (n = 435) of cows in anestrus had their ovaries evaluated using ultrasound at the time of P4 removal and at TAI. Data were analyzed by orthogonal contrasts (C): C1 (eCG effect) and C2 (eCG dose effect). Estrous occurrence (control = 53.7%, 300 IU = 70.6%, and 400 IU = 77.0%) and pregnancy per artificial insemination (control = 29.7%, 300 IU = 44.8%, and 400 IU = 47.6%) were improved by eCG treatment (C1; P = 0.0004 and P < 0.0001, respectively). Furthermore, the cows receiving eCG presented larger follicles at TAI (control = 13.5 ± 0.3 mm, 300 IU = 14.0 ± 0.2 mm, and 400 IU = 15.1 ± 0.3 mm; P < 0.0001; C1). However, there was no effect of eCG dose on any response variables studied (C2; P > 0.15). In conclusion, the eCG treatment administered at the time of P4 removal increased the occurrence of estrus, the larger follicles at TAI, and pregnancy per artificial insemination of suckled B taurus beef cows. Despite the greater occurrence of estrus in noncyclic cows receiving 400 IU of eCG, both eCG doses (300 and 400 IU) were equally efficient to improve pregnancy to artificial insemination.

Reduction of centrifugation force in discontinuous percoll gradients increases in vitro fertilization rates without reducing bovine sperm recovery.

The objective of this study was to determine the effect of different centrifugation forces in bovine sperm separation by discontinuous Percoll gradients for in vitro fertilization IVF. The semen samples from each bull were pooled or each bull were centrifuged separately and centrifuged in discontinuous Percoll gradients (30, 60 and 90%) at different forces: F1 (9000×g), F2 (6500×g), F3 (4500×g) and F4 (2200×g), according experiment. The sperm samples were evaluated to determine the concentration, motility, vigor, morphology, reactive oxygen species (ROS), integrity of the plasma membrane, lipid peroxidation, antioxidants and embryo development were also evaluated. No difference was observed in the concentration of sperm submitted to different centrifugation forces. The total percentage of motile sperm was increased after centrifugation at F3 and F4, and the ROS production at F1 was greater than the other forces. When the bulls semen were processed individually, no significant differences were observed for the sperm quality parameters between F1 and F4, including lipid peroxidation, antioxidants, cleavage rate and average time to the first cleavage. This work demonstrated for the first time that centrifugation at 2200×g enhanced the sperm penetration and fertilization rates without reducing sperm recovery compared to the typical centrifugation force (9000×g) currently used by the commercial bovine IVF industry.

Intracytoplasmic sperm injection improves in vitro embryo production from poor quality bovine oocytes.

The objective was to use subzonal sperm injection (SUZI) to understand sperm penetration patterns and to use intracytoplasmic sperm injection (ICSI) to improve production of bovine embryos using poor quality gametes. In experiment 1, poor versus good quality oocytes were fertilized with sperm from two bulls, A and B, with poor and good sperm vigor, respectively. The blastocyst rate was higher for good versus poor quality oocytes (23.3% vs. 11.1%, P < 0.05), regardless of the bull used. There was no significant difference in blastocyst rate for bull A (low vigor) regardless of oocyte quality, and for bull B (high vigor), blastocyst rate was better for good versus poor quality oocytes (25.7% vs. 9.2%, P < 0.05). In experiment 2, poor quality oocytes were subjected to SUZI. The oocyte penetration rate was lower for bull A than for bull B (29.6% vs. 53.8%, P < 0.05), when SUZI was performed within 1 hour after sperm processing. However, when SUZI was performed 2 to 3 hours after sperm processing, penetrating capacity was similar between bulls, but for bull B, penetrating capacity significantly decreased after 3 hours of sperm processing. In an attempt to overcome sperm penetrating disorders, poor and good quality oocytes were subjected to ICSI (experiment 3). Irrespective of the bull or of the oocyte quality grade, there were no differences in cleavage or blastocyst rates. Both bulls had distinct IVF embryo production rates, which we inferred were because of particular individual sperm characteristics. In conclusion, ICSI was an effective means to achieve in vitro production of bovine embryos with gametes of variable quality.

Fetal calf serum enhances in vitro production of Bos taurus indicus embryos.

The objective of this study was to determine the effect of fetal calf serum (FCS) on the quality of in vitro produced bovine embryos. Cumulus oocyte-complexes (COCs, n = 2 449) recovered by ovum pick-up from Bos taurus indicus donors were randomly assigned to experimental groups. Sperm selected by Percoll gradient was used for in vitro fertilization (insemination = Day 0). In Experiment 1 (n = 1 745 COCs), zygotes were cultured in vitro in Synthetic Oviduct Fluid + 4 mg/mL of bovine serum albumin (BSA), or BSA + 2% FCS (BSA+FCS). In Experiment 2 (n = 704 COCs), the COCs were cultured in SOF + BSA, BSA + 2% FCS, or BSA + 2% FCS on D4 (BSA + FCSD4). In Experiment 1, blastocyst yield (51%) and Quality I blastocysts (41%) at Day 7 were higher (P < 0.05) in the BSA + FCS treatment than in BSA (42 and 30%, respectively). In Experiment 2, blastocyst yield was higher (P < 0.05) in the BSA+FCS (47%) treatment. Quality I blastocyst yield was higher (P < 0.05) for BSA + FCS (34%) and BSA+FCSD4 (32%) compared to the BSA treatment (20%). A total of 820 embryos were transferred, with no significant differences among groups in pregnancy rates. In conclusion, in vitro culture in SOFaaci + BSA + FCS enhanced blastocyst yield and Quality I blastocysts; adding FCS to the culture medium increased the efficiency of IVP of bovine embryos.

Addition of recombinant human growth hormone to in vitro maturation medium of bovine oocytes.

The aim of this study was to increase the bovine embryonic development rate, adding recombinant human growth hormone (rhGH) to maturation medium of bovine oocytes. Oocytes were matured for 24 h in TCM 199 Earle's salts and five treatments were developed: T1, 0.01 IU/ml of recombinant human follicle stimulating hormone (rhFSH); T2, 0.01 IU/ml of rhFSH + 100 ng/ml of rhGH; T3, 0.01 IU/ml of rhFSH + 1000 ng/ml of rhGH; T4, 100 ng/ml of rhGH; and T5, 1000 ng/ml of rhGH at 39 degrees C and 5% of CO(2) in air and saturated humidity. In vitro fertilization from cumulus-oocyte complexes was conducted in TALP-Fert medium (18-22 h) and spermatozoa were selected by Percoll gradient. Zygotes were incubated in SOFaaci medium in 5% of CO(2) in air, 5% of O(2) at 39 degrees C and saturated humidity for 11 days. There was no statistical difference in cleavage rate and embryo production on day 7 and day 9 among treatments. However, the hatching rate increased significantly in the T4 and T5 treatments (11.0 and 12.8%, respectively), compared with the T1 treatment (4.6%) (p < 0.05). Therefore, the rhGH addition to the oocyte maturation medium showed beneficial effects on the hatching rate of in vitro-produced bovine embryos.