Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos.

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.

Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blastocysts derived in vitro or in vivo.

Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO2 production from (U-14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.

Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo.

This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity.

Plasma progesterone profiles and fertility status of anestrus Zebu cattle treated with norgestomet-estradiol-eCG regimen.

Zebu cattle are notorious for poor fertility characterized by late maturity and long intercalving intervals attributed to a variety of factors, including genetic, nutritional and climatic. The aim of the present investigation, therefore, was to induce fertile estrus in acyclic pubertal heifers and postpartum anestrous Zebu cows by hormonal intervention. Pubertal Hariana and Sahiwal anestrous heifers (n = 51) and postpartum cows (n = 55) were either assigned a placebo (controls, n = 6 for each breed and parity) or treated with 10-d norgestomet (3 mg) subcutaneous ear implants, with an initial injection of 3 mg, i.m. norgestomet + 5 mg estradiol valerate, followed by 500 IU eCG at implant withdrawal (NOR-treated groups). Jugular venous plasma samples were obtained from a total of 28 animals (controls: 4 heifers and 4 cows; NOR-treated: 12 heifers and 8 cows) on Days 0 (implant insertion), 3, 7, 9 and Day 10 (implant withdrawal), every 12 h on Days 11 and 12, and then once daily on Days 17, 24 and 31. All the samples were assayed for progesterone. Almost all (97%) heifers and 81% cows were induced to estrus, the majority (92% heifers and 79% cows) within 120 h of implant removal. Synchrony of the induced estrus was better in cows, but interval to estrus and estrus duration were significantly longer in heifers (P < 0.05). Post-treatment fertility, based on Day 28 nonreturn rate, first service, and overall conception rates, was better in heifers (78.9, 60.5 and 73.7%, respectively) than cows (77.1, 48.6 and 62.9%, respectively), but the differences were significant only for the overall pregnancy rate (71.8% for heifers and 51.2% for cows; P < 0.05). Low pre-treatment plasma progesterone values (< 0.5 ng/mL) were consistent with ovarian inactivity, confirming the true anestrus status of experimental animals. Controls failed to exhibit estrus and maintained low progesterone concentrations throughout the study. In treated animals, high progesterone values from Day 17 onwards suggested ovulatory estrus. These early luteal phase progesterone concentrations in nonpregnant (P = 0.06) and nonpregnant, nonreturn (P < 0.05) animals were low in comparison with those of pregnant animals. Good fertility resulting from breeding according to estrus, inspite of variable intervals to estrus and estrus duration, advocates its advantage over fixed-time insemination in norgestomet-treated anestrous Zebu cattle.

Effects of macromolecules recovered from uterine luminal fluid on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

Day-4 mouse embryos grew well in culture media supplemented with macromolecular components of uterine fluids recovered on day 3, 4 or 5 of pregnancy and pseudopregnancy. Addition of these components to media during a 2-h pulse culture had no significant effect on the incorporation of glucose carbon by morulae/early blastocysts. However, various fractions of uterine luminal macro-molecules significantly increased the turnover of glucose carbon incorporated into acid-soluble and acid-insoluble glycogen, into nucleic acids and into proteins during a 24-h chase culture. These effects were due mainly to components with a molecular weight between 1000 and 10,000 Da and the activity was most marked in fluids collected on day 5 of pregnancy or pseudopregnancy. Oxidation of glucose during a 4-h incubation was inhibited in the presence of certain uterine macromolecules but most consistently by the large molecular weight component (greater than 300,000 Da). Some differences were noted in the inhibitory activity of macromolecules obtained from pregnant and pseudopregnant sources. There was little evidence of an effect of uterine-fluid components on lactate production from glucose.

Effects of oxygen concentration on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

The utilization of the acid-soluble glycogen pool in pulse-labelled embryos was significantly enhanced during 24- and 48-h chase culture under low oxygen concentrations of 5, 2.5 and 1%. The lower the oxygen tension the greater was the turnover in the pool. The morphological development of embryos was equally as good at very low oxygen concentrations as when embryos were cultured in 5-20% oxygen. Reduction in oxygen concentration enhanced the oxidative utilization of substrate, as measured by rate of carbon dioxide production. The present study could provide an explanation for the discrepancy in glycogen content between mouse blastocysts developing in utero and in vitro and for the reported beneficial effects of low oxygen concentration during development of embryos in culture.

Effects of oestradiol and progesterone on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

The addition of progesterone (10(-7) to 10(-5) M) and/or oestradiol (10(-10) M) during 24-h chase culture of pulse-labelled morulae-early blastocysts did not affect the degradation of radiolabelled glycogen or other biochemical fractions. The presence of a high concentration of progesterone (10(-5) M) during 5-h pulse culture significantly inhibited incorporation of substrate carbon from [U-14C]glucose into both the acid-soluble and acid-insoluble glycogen fractions, but had no effect on non-glycogen fractions. Catabolic utilization of glucose as estimated by the rate of carbon dioxide and lactate production was not affected by the presence of progesterone (10(-7) to 10(-5) M), oestradiol (10(-10) to 10(-8) M) or a combination of both. The results indicate that ovarian steroids at expected physiological concentrations do not directly influence embryonic energy metabolism.

Effects of coculture with uterine epithelial cells on the metabolism of glucose by mouse morulae and early blastocysts.

Coculture of mouse morulae/early blastocysts with isolated endometrial epithelial cells reduced incorporation of glucose carbon into embryonic glycogen but had no significant effect on incorporation into other internal carbon pools during a 5-h culture in serum-supplemented Dulbecco's modification of Eagle's minimum essential medium. Turnover of glycogen pools during 24-h chase culture of pulse-labelled embryos was unaffected by the presence of uterine epithelial cells recovered from day-4 pregnant or non-pregnant mice. However, significantly more label was retained in non-glycogen macromolecules during chase in the presence of endometrium recovered from non-pregnant than from pregnant uteri.

Effects of prostaglandins E-2 and F-2 alpha on the metabolism of [U-14C]glucose by mouse morulae-early blastocysts in vitro.

During 5-h culture in the presence of radioactive glucose, PGE-2 (10 micrograms/ml) significantly inhibited incorporation of glucose into the acid-soluble glycogen pool. PGE-2 at 1 and 10 micrograms/ml and PGF-2 alpha at 1 microgram but not 10 micrograms/ml stimulated incorporation of glucose into non-glycogen macromolecules during culture. However, the utilization of acid-soluble glycogen and other biochemical pools was not affected by the presence of PGs in the medium during 24-h chase culture of pulse-labelled embryos. Carbon dioxide production was significantly suppressed in the presence of PGs but accumulation of lactate was not affected. The results indicate that PGE-2 and PGF-2 alpha, in physiological concentrations, directly influence the metabolism of glucose by preimplantation embryos.

Metabolism of glucose by preimplantation mouse embryos in the presence of glucagon, insulin, epinephrine, cAMP, theophylline and caffeine.

Neither insulin nor epinephrine influenced the incorporation of glucose into the acid-soluble or acid-insoluble glycogen pool of mouse embryos at the morula-early blastocyst stage during 5 h culture in the presence of radiolabelled glucose. During a 5 h chase culture of pulse-labelled embryos at this stage of development, acid-soluble glycogen labelled during the pulse was not utilized by the embryo but acid-insoluble glycogen was reduced. Addition of glucagon, insulin, epinephrine, cAMP, theophylline or caffeine during chase culture had no effect on the turnover of label in the glycogen pools of the embryo. These results indicate that the turnover of embryonic glycogen observed in vivo is not due to the direct effect of the hormones that regulate glycogen metabolism in the mother. Insulin was found to stimulate incorporation of glucose into non-glycogen macromolecules during both pulse and chase culture. Thus, whilst an effect of insulin on glycogen metabolism was absent, the anabolic effects of this hormone appear to have been expressed in the embryo at this stage of development.

Pregnancy in suboestrus buffaloes (Bubalus bubalis) after treatment with Prostaglandin F(2) alpha.

Seventy six apparently anoestrous buffaloes, with a palpable corpus luteum at day 43 to 547 post partum, were treated intramuscularly with Prostaglandin, using either 25 mg single dose (Group I), 25 mg double dose 11 days apart (Group II), or 50 mg single dose (Group III). Animals exhibiting oestrus, without any treatment, during the period of experimentation served as control (Group IV). The overall conception rate for the treated animals was 28.8, 51,5, and 69.7 percents after one, two, and three inseminations, respectively. The conception rate, at induced as well as subsequent oestrus, was comparable to control animals. The intergroup differences among treated animals were not significant. The conception rate was greater when the induced heat was more intensely expressed. The subsequent mean oestrus cycle length was similar to controls when all of the treated animals were considered together.