PH

The pH of fluid collected from the reproductive tract of rhesus monkey was reported to alter in parallel with changes in the bicarbonate concentration increasing from 7.1 to 7.3 during the follicular phase of the estrous cycle to 7.5-8.0 at the time of ovulation and during luteal phase (272). Dale et al. (273) recently reported that the pH of the uterine fluid is lower than that of the oviduct. External pH of culture media formulated for preimplanta-tion embryos is commonly between 7.3 and 7.4 (depending on the CO2 concentration used). Studies on the mouse embryo determined that development from the 2-cell stage to the blastocyst stage could occur in media with a pH range from 5.9 to 7.8 (65). Similarly, hamster 8-cell embryos could develop to the blastocyst stage in medium with an external pH range from 6.4 to 7.4 (269). In contrast however, it has recently been shown that a transient exposure of zygotes and 2-cell mouse embryos to medium with elevated pH significantly reduced subsequent development to the blastocyst stage (274). Although these studies demonstrated that some embryos could still develop to the blastocyst stage in a wide range of external pH's, the subsequent viability of these embryos following transfer is unknown. Subsequently, there has been considerable work in the area of intracellu-lar pH (pHi) and its role in regulating embryo development. It has been established that even relatively small fluctuations in pHi can significantly retard subsequent developmental competence. Fluctuations in either the acidic (84,275) or the alkaline (276) range can drastically reduce development. Even more significantly, it has been determined that mammalian oocytes and embryos for around six hours following fertilization lack any functional transport systems to regulate pHi in either the acid (275) or the alkaline (275,277) ranges. Therefore, care should be taken to avoid fluctuations in the pH of media during embryo manipulation and culture. This is especially relevant for oocytes that are stripped of their cumulus before an ICSI procedure. Immediately following the denudation procedure these oocytes and embryos cannot regulate their ionic homeostasis. Amino acids are also known to increase the intrinsic buffering capacity of the embryo and reduce fluctuations in intracellular pH of the embryo. Therefore, as discussed earlier, addition of amino acids to the culture or handling medium

Table 7 Preparation of Color Standards for pH of Media pH at 18°C Solution A (mL) Solution B (mL)

Table 7 Preparation of Color Standards for pH of Media pH at 18°C Solution A (mL) Solution B (mL)

6.6

62.7

37.3

6.8

50.8

49.2

7.0

39.2

60.8

7.2

28.5

71.5

7.4

19.6

80.4

7.6

13.2

86.8

Note: Stock A: 9.08g KH2PO4 (0.067M), 10mg phenol red in 1L of water. Stock B: 9.46g Na2HPO4 (0.067 M), 10mg phenol red in 1 L of water. Measure the pH with meter, and adjust pH as required, i.e., add solution A to lower the pH (make more acidic), add solution B to increase the pH (make more alkaline). The pH standards should be filter-sterilized and can then be kept for up to six months.

Note: Stock A: 9.08g KH2PO4 (0.067M), 10mg phenol red in 1L of water. Stock B: 9.46g Na2HPO4 (0.067 M), 10mg phenol red in 1 L of water. Measure the pH with meter, and adjust pH as required, i.e., add solution A to lower the pH (make more acidic), add solution B to increase the pH (make more alkaline). The pH standards should be filter-sterilized and can then be kept for up to six months.

can also help to reduce pHi fluctuations (31). It is also important to consider the relationship between the pH of the culture medium (pHo) and the pHi of the embryo; over a pHo range of 7.0-7.6, the pHi of the mouse zygote remained at 7.17. Rather than pHo affecting pHi directly, the presence of weak acids or bases in the culture medium dramatically affects pHi (84).

The pH of a CO2/bicarbonate buffered medium is not easy to quan-titate. A pH electrode can be used, but one must be quick and the same technician must take all readings to ensure consistency. Solid state probes are now available with a higher degree of accuracy. An alternative approach is to take samples of media and measure the pH with a blood-gas analyzer. A simple and reliable method of checking the pH of a medium is to use color standards. The color standards use 0.067 M solutions of potassium phosphate and sodium phosphate. These solutions are then added together in varying quantities to produce solutions of the required pH. The preparation of such standards is shown in Table 7. However, this approach requires the medium to contain phenol red.

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Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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