296 EFFECT OF FROZEN SEMEN WITH OSTEOPONTIN ON IN VITRO BOVINE FERTILIZATION AND EMBRYO DEVELOPMENT

Abstract

Poor reproductive performance of dairy cattle has a major negative impact on farm profitability. This is in part attributed to the use of semen from bulls of below-average fertility and to early embryonic mortality. Osteopontin (OPN) is an acidic glycoprotein that has been detected in the epithelium of the ampulla, seminal vesicles, and seminal fluid of high-fertility Holstein bulls. The objective of this study was to determine whether freezing semen with purified bovine milk OPN would improve fertilization and embryonic development in vitro. In a first step, frozen semen from 6 bulls was used with different concentrations of OPN (0, 1, 10, and 100 µg mL^-1) to evaluate fertilization, and in a second step, semen from 2 of the 6 bulls was frozen with OPN (0, 5, 10, 20, 40, and 80 µg mL^-1) to evaluate its effect on in vitro fertilization and embryonic development. In vitro-matured bovine oocytes were inseminated with 1 × 10⁵ frozen–thawed spermatozoa per 10 oocytes. After 18 h (39°C, 5% CO₂ in air), oocytes designated for evaluation of fertilization were fixed, stained, and observed to determine the presence of pronuclei. Oocytes destined for embryo culture were placed in 4-well dishes containing 500 µL of synthetic oviduct fluid per well at 5% O₂, 5% CO₂, and 90% N₂ (v/v). Each experiment was repeated 4 times, and data from each experiment were pooled. Analysis of variance using a general linear model was performed using a weighted mean based on the number of oocytes per treatment. Significantly more (P < 0.05) oocytes were fertilized when frozen semen was used with 10 µg mL^-1 of OPN (85 ± 4.0%, 78 ± 4.0%) from 2 of the 6 bulls than when 0 µg mL^-1 was used (75 ± 4.0%, 69 ± 4.0%). Semen from those bulls resultewd in more fertilized oocytes when 20 µg mL^-1 (86 ± 3.5%, 79 ± 3.4%) and 40 µg mL^-1 (88 ± 3.4%, 81 ± 3.9%) of OPN were used during the second phase of the experiment. Oocytes inseminated with 10 µg mL^-1 (84 ± 2.5%, 77 ± 2.3%), 20 µg mL^-1 (87 ± 2.8%, 79 ± 1.9%), or 40 µg mL^-1 (89 ± 1.9%, 81 ± 2.4%) of OPN in the frozen semen had increased cleavage rates at Day 4 compared with those inseminated with 0 µg mL^-1 (75 ± 3.5%, 69 ± 3.4%). At Day 8, blastocyst development was greater for 10 µg mL^-1 (40 ± 1.8%, 37 ± 1.6%), 20 µg mL^-1 (42 ± 2.1%, 38 ± 2.9%), and 40 µg mL^-1 (45 ± 2.9%, 40 ± 2.5%) of OPN than for the semen with 0 µg mL^-1 (33 ± 2.3%, 29 ± 2.8%). We conclude that semen from some bulls that was frozen with purified bovine milk OPN increases IVF, cleavage, and embryonic development, suggesting a facilitative role for OPN in some reproductive technologies.