Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos

Abstract

The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UF) on Day 3 (D3) and Day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 non-protein amino acids (β Alanine, Taurine, Ornithine and Citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.06±3.63 mmol/L), D3UF (10.54±5.16 mmol/L) or D5UF (10.23±6.69 mmol/L). But the total concentration of amino acids in D5OF (5.89±1.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected from and the day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.3±7.9%) or D5UF (PZM3-D5UF, 14.3±10.7%) concentrations or in PZM3-D3OF for the first 48 (20.5±15.1), 72 (25.6 ±10.4) and 96 (18.7±10.0) hours and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.8±9.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.6±8.3) was increased compared to PZM3-SAA (40.5±3.5). The osmolalities in D3OF, D3UF, D5OF and D5UF were 318±8, 320±32, 321 and 293±8 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150-360mOsM), higher blastocyst rates were obtained at 270-300 mOsM in the PZM3-SAA group (24.6%-33.9%) and 270-290 mOsM in PZM3-D3OF group (22.4%-24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hours and then transferred to 250 mOsM (33.3±3.4%), the blastocyst rate was higher than original PZM3 (21.2±2.2%)(288 mOsM).

A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos

“…IVP techniques are particularly important in livestock breeding because embryo manipulation is directly related to economic aspects of the agricultural industry [4]. To improve the developmental efficiency and quality of embryos, many studies have addressed the chemical modification of medium [5] and its components, as well as environmental changes in physiological conditions, such as temperature [6], pH, shear stress [7], and osmolality [8]. Although these efforts have revealed many factors that are critical to developing embryos, the development rate of embryos, especially that of large animals such as porcine and bovine species, still remains low [9].…”

Localisation and function of voltage-dependent anion channels (VDAC) in bovine spermatozoa

“…To investigate the volumetric behaviour and to test the hypothesis whether VDAC may be involved in the control of sperm cell volume and prevention of its disturbance, we evaluated the effect of anti-VDAC antibodies on sperm capability to maintain cell volume at 300 mOsmol/kg. These conditions are close to the osmolality of fluids of female genital tract reported for many mammalian species (280-320 mOsmol/kg, [7,8,28]) and to the osmolality of bovine seminal plasma, which is slightly higher (340 mOsmol/kg, [38]). …”

Cell volume regulation in mammalian oocytes and preimplantation embryos

“…This proved unlikely, however, since determinations of mouse oviductal fluid osmolality indicated that it was in the 300-310 mOsM range (Collins and Baltz, 1999;Fiorenza et al, 2004). Similarly, porcine oviductal fluid is reportedly $320 mOsM (Li et al, 2007) while that of rat is $290 mOsM (Waring, 1976). Each species' oviductal fluid is thus essentially isoosmotic to blood in the same species (Waymouth, 1970;Williams et al, 1972;Knudsen et al, 1979) and thus not hypotonic, like the newer culture media had been.…”