Electrolytes, Metabolic And Acid-Base Parameters In Blood And Fluids of The Reproductive Tract During In Vivo Maturation of Bovine Oocytes

Using Abbott iSTAT1™ and NanoDrop, we determined the dynamics of acid-base, electrolyte, 19 metabolites, and total protein in venous blood, fluids of the dominant follicle (FF), oviduct (OF), 20 and uterus (UF) during the window of oocyte maturation. Holstein heifers (n=36) were 21 synchronized with PGF 2 α on Days -11 and 0, CIDR during Days -6 to 1, and GnRH given on Day 22 2 after 2 nd PG. Samples were collected at 24h, 48h, 60h, 72h, and 78h after 2 nd PG. Most 23 electrolytes analyzed, Cl - , K + , and Ca 2+ were significantly affected in blood and FF ( P <0.05) by 24 CIDR removal. Similarly, Cl - and Na + also significantly changed in OF and UF across time. 25 Glucose, lactate, and significantly changed across O during 29 vitro conclusion, components the fluid undergo major changes during the


INTRODUCTION
Therefore, the determination of the in vivo microenvironments supporting oocyte Numerous prior studies have reported electrolytes, metabolic and acid-base parameters in 55 bovine blood and follicular and a few on oviductal, and uterine fluids 8,9,12 . However, the vast 56 majority of them used slaughterhouse materials 13 . Among the few that did conduct a controlled 57 study with clearly defined reproductive status, samples were taken from different days of the 58 estrous cycle. No reports to date followed the progression of changes in these fluids during the 59 specific window of time in which the oocytes mature in vivo. 60 The Abbott point-of-care handheld blood analyzer, iSTAT, is a convenient device for 61 measurements of tissue fluids. A few recent studies have reported its adaption in the bovine blood 62 and tissue fluids 14,15,16 . In this study, we collected serum, follicular, oviductal, and uterine fluids 63 in Holstein heifers 24h before and after the LH surge, during which the follicles undergo final 64 development, and the oocyte matures. The dynamics of components of these fluids were 65 determined by iSTAT. We found significant changes in most parameters studied. The data 66 obtained here are good references to improve the in vitro oocyte maturation conditions. 67 69 The present study was approved by the Institutional Animal Care and Use Committee  releasing hormone (GnRH; 2 ml of Factrel injection, 50 μg/ml) was given intramuscularly to 86 induce the LH surge. This protocol ensures tight estrus synchronization through rapid progesterone 87 decline (after CIDR removal) and precise LH surge (induced by GnRH).

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After quarantine, jugular venous blood samples were collected daily into silicone-coated 89 vacutainer tubes without anti-coagulants. The samples were allowed to clot and then centrifuged 90 at 2,500g for 15 min in a refrigerated centrifuge (4°C). The serum was stored at -80°C until 91 analysis. Daily ultrasound of the ovaries using an Aloka 500 ultrasound machine equipped with a 92 linear probe of 5 MHz was conducted starting from the second set of PGF2α injections (Day 0).

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To avoid manual ovulation, ultrasound was not conducted on the day of slaughter.

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The animals were sacrificed at 24, 48, 60, 72, and 78 h after the first PGF2α injection of 95 the second set (Day 0), and reproductive tracts were transported at ambient temperature to the lab 96 for further processing. To collect fluids from the oviduct and uterus, the uterotubal junction and the uterine- The NanoDrop 1000™ was used to measure the total protein in both the serum and FF.

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The CHEM8+ determines sodium (Na + ), potassium (K + ), chloride (Cl -), ionized calcium (iCa 2+ ), 123 TCO2, glucose, urea nitrogen (BUN)/urea, creatinine, hematocrit, and hemoglobin. The iSTAT 124 device was designed for use with human blood but has been used off-label in other species and 125 tissue fluids 15,16 . The caveat of this off-label use is that the cartridges were calibrated to the ranges 126 of parameters in humans. Animal fluids may contain levels higher or lower than the maximum or

Statistical analysis
We used R © software 21 to run one-way ANOVA across different time points after PGF2α    151 Changes in the microenvironment of oocytes during the periods of final oocyte 152 development and resumption of meiosis were monitored in follicular fluids between ~26 h before 153 and 30 h after the LH surge, respectively. Jugular venous blood is much easier to collected and 154 used to reveal whether blood parameters could predict the follicular microenvironment.

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While the FF is mainly derived from the blood serum, most parameters were very different 156 between these two fluids. For example, total protein concentrations in serum averaged 87.80 ± 157 9.50 mg/ml and was unaffected by luteolysis ( Fig. 3). This was expected as serum protein levels 158 should not dramatically change according to ovarian development. Follicular fluid, however, 159 contained a much less total protein with an overall mean of 51.00 ± 12.11 mg/ml. Furthermore,   Three out of four electrolytes (Cl -, K + and Ca 2+ ) have significantly changed in blood with 164 P-value of 0.037, 0.014, and 0.0008, respectively, but Na + with P-value of 0.079 was not 165 significant. Surprisingly, FF parameters (K + , Na + , and Ca 2+ ) did not change significantly across 166 time with P-value of 0.099, 0.079, and 0.096, respectively, and Clwas not significant with P-167 value of 0.143 (Fig. 4). Interestingly all electrolytes in FF underwent major changes between 24 168 and 48 h (pairwise comparison P<0.05) after PGF2α when luteolysis/CIDR removal occurred 169 which should cause a drastic and rapid decline in progesterone levels, but not significant changes 170 across all time points. In FF, Cland K + significantly decreased, while Ca 2+ and Na + increased.

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Most electrolytes in FF, however, attempted to return to pre-luteolysis levels by 60h post-PGF2α.   As expected, the blood pH was well-maintained across treatment times (Fig. 6a). However, 186 the pH in FF (Fig. 6b) significantly decreased as follicles approached ovulation (P=0.028). Blood 187 glucose, lactate, and creatinine also did not significantly change across time (Fig. 6a). Significant One of the objectives of this study was to determine if blood parameters could be used to 200 estimate changes in FF. We calculated the correlations for nine parameters (Fig. 7) and found that  parameters. The number of samples that gave successful readings is included in Table 1  oviductal Cl -(P=0.002), TCO2 (P=0.005) and glucose (P=0.049); (Fig. 8), as well as uterine Na +

Differences across tissue fluids 217
Although a parameter may vary significantly across time points within a tissue fluid, the 218 differences of a particular parameter across fluids were even greater (Fig. 9). To demonstrate these 219 differences, we pooled data within tissue fluids across time points. While Na + and Clwere 220 relatively similar across the four different fluids, K + , urea nitrogen, and creatinine were drastically 221 different. Specifically, K + (Fig. 9b) was the highest in OF and UF. It is worthy to mention that the 222 iSTAT has a detection limit for K + at 9 mM and multiple measurements for OF and UF were out 223 of range, showing readings as >9 mM, and puzzlingly, still unattainable after dilutions. Therefore, 224 the K + levels in these two fluids could be much higher than 9.0 mM. Conversely, the iCa 2+ was the 225 lowest in UF (Fig. 9b). Urea nitrogen and creatinine (Fig. 9c) were higher in OF than both blood 226 and FF and highest in UF, likely a result of differences in cellular activities. Levels (+SEM) of electrolytes and metabolites in the four tissue fluids. a) Na + and Clwere relatively similar among fluids. b) K + is higher in FF than blood and highest in OF and UF. The* is donated to the maximal reading of iSTAT (K + >9.0 mM) was reached in both OF and UF. The iCa 2+ was lowest in UF. c) Urea Nitrogen was similar in blood and FF but higher in OF and highest in UF. Creatinine was higher in OF and UF than FF and blood. When examining the pooled data on gases (Fig. 10), we found that in FF the pCO2 and 236 pO2was approximately 5X and 2X higher than the levels in venous blood, respectively. These Hugentobler et al. 31 reported striking higher K + and lower Cland Na + on Day 14 than other days.

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None of these prior studies, however, were designed to capture tissue fluid changes during the 256 window of in vivo oocyte maturation.

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In all tissue fluids studied, Na + and Clare the major electrolytes and appear to be maintained at 260 comparable levels to preserve a charge-neutral environment. These two electrolytes also varied the 261 least from one fluid to another. For example, Clonly varied 0.005, 3, and 11%, respectively, from 262 blood to OF, FF, and UF. The Na + levels in the three tissue fluids also stayed close to those in the 263 blood and FF, varying less than 15% in UF and OF. The most varied electrolyte was K + , which was high in both UF or OF and was more than 52% and 24% lower in blood and FF, respectively.

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It is known that K + levels in the blood are tightly regulated through kidney excretion, and the 266 concentration gradient for K + between serum and FF suggests an active inward transport 27 . The  4. Off-label use of iSTAT in tissue fluids other than the human blood 338 The iSTAT has been validated for the determination of pH, pCO2, pO2, total CO2, oxygen 339 saturation, base excess, HCO3 -, Na + , K + , ionized Ca 2+ , hematocrit, and hemoglobin  It is worthy to mention that we also tried to use these cartridges of the iSTAT in uterine 347 and oviductal fluids but many results, such as pO2, pCO2, HCO3 -, and other parameters from CG4+ 348 cartridge, could not be obtained, likely due to interference by cellular debris in these samples. Most 349 samples were so thick that even we inverted the cartridges to allow gravity to help the sample pass 350 through the narrow capillary channel, the instrument still failed to produce readings. Other measurement failures were caused by out-of-range levels. The iSTAT is designed for human blood 352 and the detection ranges were optimized for this sole purpose. For example, the maximum 353 detection limit for K + is 9 mM. However, multiple oviductal and uterine fluids gave the reading of 354 >9 mM. Strangely enough, diluting the samples with deionized water did not solve the problem.

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The previously reported oviductal and uterine K + levels were 6 and 9-13 mM, respectively 31 . While