Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre‐exposure of oocytes to Ricinus communis (RCA‐1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm–egg binding and fertilization. In vitro‐matured bovine oocytes were incubated (39°C, 5% CO₂ in air) for 2 h in the following treatments: (i) 500 μl of fertilization medium (FM); (ii) 250 μl of FM with 0.25 ml of non‐luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 μl of FM with 250 μl of NLAUTF and 4 μl of RCA‐1 lectin; (iv) 250 μl of FM with 250 μl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA‐1 lectin; (v) 500 μl of FM and RCA‐1 lectin. Following incubation, oocytes were washed, placed in FM with 2 μg heparin, and incubated with 1 × 10⁵ frozen–thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate‐orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean ± SEM) when oocytes were incubated in treatment 3 (59.0 ± 5.5) than in treatments 2 (46.4 ± 5.6), 4 (18.1 ± 5.4), 5 (33.4 ± 5.6) or 1 (32.5 ± 5.6). More oocytes were fertilized when incubated in treatment 3 (91% ± 3.0) than in 2 (84% ± 3.0), 4 (40% ± 3.0), 5 (77% ± 3.0) or 1 (76% ± 3.0). As in previous studies, this study suggests that RCA‐1 lectin enhances binding of UTF‐derived OPN to bovine oocytes, resulting in increased sperm–egg binding and fertilization in vitro and a possible role in fertilization.     

Hepatic steroid inactivating enzymes,hepatic portal blood flow and corpus luteum blood perfusion in cattle

Production from the corpus luteum (CL) and/or hepatic steroid inactivation impacts peripheral concentrations of P4, which can alter reproductive performance. Our primary objective was to examine hepatic steroid inactivating enzymes, portal blood flow, and luteal blood perfusion at 10 days post‐insemination in pregnant versus non‐pregnant beef and dairy cows. Twenty early lactation Holstein cows and 20 lactating commercial beef cows were utilized for this study. At day 10 post‐insemination, hepatic portal blood flow and CL blood perfusion were measured via Doppler ultrasonography. Liver biopsies were collected and frozen for later determination of cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A), uridine diphosphate‐glucuronosyltransferase (UGT) and aldo‐keto reductase 1C (AKR1C) activities. Pregnancy was determined at day 30 post‐insemination and treatment groups were retrospectively assigned as pregnant or non‐pregnant. Data were analyzed using the mixed procedure of SAS. Steroid metabolizing enzyme activity was not different (p > .10) between pregnant versus non‐pregnant beef or dairy cows. Hepatic portal blood flow tended (p < .10) to be increased in pregnant versus non‐pregnant dairy cows. Luteal blood perfusion was increased (p < .05) in pregnant versus non‐pregnant dairy cows. Pregnant dairy cows appear to have an increased rate of hepatic clearance of P4 in combination with increased synthesis from the CL. This could account for the lack of difference in peripheral P4 concentrations between pregnant and non‐pregnant dairy cows. This study highlights the relevance of further investigation into steroid secretion and inactivation and their impact on the maintenance of pregnancy in cattle.