High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins.

In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins

Follicular size, follicular atresia, and oocyte morphology were investigated for the possible relation of these characteristics to the developmental competence of bovine oocytes. Ovaries from a local slaughterhouse were dissected to obtain a heterogeneous population of follicles. Half of each follicle was fixed for histological analysis, and the oocytes were detached carefully and cultured individually. Before in vitro maturation, the oocytes were grouped into six different classes based on the morphology of the cumulus and the ooplasm: classes 1 and 2 represent oocytes with a homogeneous ooplasm plus a compact and complete cumulus, and classes 3-6 represent oocytes with a granulated ooplasm and an incomplete and/or expanded cumulus. Oocytes from class 3 (beginning of expansion in outer cumulus layers and slight granulations in the ooplasm) developed past the 16-cell stage significantly (P < 0.05) more than oocytes with a compact and complete cumulus (classes 1 and 2) and oocytes from classes 4-6 (incomplete and/or expanded cumulus) after 5 days of in vitro culture. Oocytes from follicles measuring 3 mm or less did not develop past the 16-cell stage, whereas follicles of 3-5 mm and 5 mm or larger developed at similar rates (17% and 21% morulae, respectively). The state of the follicle did not affect whether an embryo reached at least the 16-cell stage, as comparable rates were obtained in all three groups of follicles: nonatretic (20%), intermediate (14%), and slightly atretic (16%).(ABSTRACT TRUNCATED AT 250 WORDS)

Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes

Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development.

Simple media were developed to study the metabolic requirements of bovine embryos up to Day 7 (Day 0 = day of oocyte aspiration) in vitro. Embryos were derived from oocytes matured and fertilized in vitro. At 45 +/- 2 h post insemination, embryos (> or = 2 cells) were randomly allotted to treatments. Examined in experiments 1 and 3 was the effect of pyruvate concentration in the presence of lactate. In the presence of lactate, pyruvate (0.2-5.0 mM) had no effect (p > 0.05) on the percentage of morulae or blastocysts. However, increasing the concentration of hemicalcium L-lactate from 5 mM to 10 mM decreased (p 0.05) on embryo development (experiment 4). However, the interaction of adding calcium and lactate resulted in a decreased (p < 0.05) percentage of morulae. Determined in experiment 6 were the independent effects of pyruvate, lactate, and glucose on embryo development in vitro. As pyruvate or lactate level was increased from 1 to 10 mM, the percentage of blastocysts was decreased (p < 0.05). These experiments indicate that adding pyruvate to a medium containing lactate is not necessary for development of bovine embryos in vitro.

Development of bovine embryos in vitro as affected by energy substrates.

In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS)

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Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium.

Oviduct tissue-conditioned medium was evaluated for the culture of IVM-IVF bovine zygotes to the compact morula (cM) and blastocyst (BL) stages. Development was unaffected (P greater than 0.50) by freezing and thawing of conditioned medium: no. cM + BL/no. cleaved ova obtained after culture in nonfrozen, frozen-thawed, and control treatments were 31/148 (21%), 26/124 (21%) and 5/86 (6%), respectively. The greatest proportion of normal development was obtained after a conditioning period of 48 h (P less than 0.05): no. of cM + BL/no. cleaved ova in media conditioned for 5, 24, 48 and 96 h were 23/114 (20%), 38/112 (34%), 43/115 (37%) and 36/125 (29%), respectively. Development declined with increasing dilutions of conditioned media (P less than 0.005): no. cM + BL/no. cleaved ova for 100, 75, 50, 25 and 0% conditioned medium were 32/94 (34%), 29/94 (31%), 17/82 (21%), 10/94 (11%) and 11/73 (15%), respectively. The oestrous cycle stage from which oviducal tissue was obtained did not affect development (P greater than 0.75); no. cM + BL/no. cleaved ova was 21/63 (33%) at oestrus and 21/79 (27%) in the luteal phase.

/pdf/some-factors-affecting-the-efficacy-of-oviduct-tissue-12l0t6w7as.pdf

W. H. Eyestone

Papers

Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium.

Some factors affecting the efficacy of oviduct tissue-conditioned medium for the culture of early bovine embryos.