PDF《Theriogenology》

5 mL/dose) was from Phoenix Pharmaceutical Inc. (St. Joseph, MO, USA). Embryo ?lters (MiniFlush Embryo System), y-tubing (Y-Junction Tubing), catheters (Silicon ET catheter CH16/CH18, two-way Foley,

30 mL balloon), me- dium for embryo recovery (BoviPro Recovery Medium with PVA, 2L), holding medium (BoviPro Holding Medium, with BSA), ethylene glycol (BoviPro Ethylene Glycol with Su- crose), embryo straws (MiniStraw, 0.25 mL), and plugs were from Minitube of America Inc. (Verona, WI, USA). The RIA kit (Coat-A-Count) was from Diagnostic Products Cor- poration (Los Angeles, CA, USA). Uterine smears were stained with Diff-Quick/Wright-Giemsa (Dade Behring, Newark, DE, USA). Nonsexed frozen semen (20 ?

106 sperm/straw) from

8 and

11 sires from several AI companies and of proven fertility (high estimated relative conception rate [ERCR]) were used in experiments

1 and 2, respectively. In experi- ment 3, all breeding were performed with nonsexed frozen semen (20 ?

106 sperm/straw) from two Holstein sires with high genetic merit, proven outstanding ?eld fertility (sire conception rate [SCR] USDA scores ! 1.5), and produced from a single ejaculate/sire. Sperm motility (sire

1 ? 53% and sire

2 ? 56% at

0 hour) was performed objectively by a computer-assisted semen analyzer (CEROS, Hamilton Thorne). Sperm abnormalities (sire

1 ? 9% and sire

2 ? 14%) and intact acrosome (sire

1 ? 61% and sire

2 ? 56%) were evaluated under differential interference contrast optics (? 600). 2.2. Animals and treatment protocol All procedures were approved by the Animal Care Committee of the College of Agriculture and Life Sciences, University of Wisconsin-Madison. Experiment 1dSeventeen nulliparous Holstein heifers (12C16 months old) were housed at the Dairy Cattle Research Center (UW-Madison, WI, USA) in loose housing with headlocks. Heifers were fed ad libitum a total mixed ration (TMR) once a day consisting of corn silage, alfalfa silage, and grass hay, supplemented with vitamins and minerals, and balanced to meet or exceed the minimum requirements. For superstimulation, heifers on Day

7 of a cycle received two PGF2a treatments,

12 hours apart, and a GnRH treatment at

24 hours after the ?rst PGF2a treatment to synchronize ovulation. A CIDR device was inserted

6 days after GnRH, and

2 days later,

10 decreasing doses of FSH were given at 12-hours intervals for

5 days with a total equivalent of

150 or

300 mg NIH-FSH-P1 (half of the ani- mals in each group were treated with either the higher or lower dose of FSH). Treatment with PGF2a was done at the seventh FSH treatment and the CIDR was removed at the time of the ninth FSH treatment. Twelve hours after the last FSH, ovulation was induced with GnRH, and

18 hours after GnRH, one dose of semen was deposited in the body of the uterus or in the greater curvature of uterine horns (half dose in each horn). All heifers were inseminated, and em- bryos were recovered by the same technician (R. Sartori). P.D. Carvalho et al. / Theriogenology

80 (2013) 1074C1081

1075 Seven days after GnRH, heifers had ova/embryos recovered using a nonsurgical shallow uterine horn technique as described by Sartori et al. [31], and each uterine horn was ?ushed separately with

1 L of embryo recovery medium. Structures were collected into an embryo ?lter, immedi- ately washed into a petri dish, searched under a stereo microscope, and classi?ed for quality (1 ? excellent,

2 ? good,

3 ? fair,

4 ? poor, and

5 ? degenerate) as previously described [32,33]. Embryos graded as 1, 2, and