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Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows
Animal Reproduction Science 80 (2004) 201–211
Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows J.L.M.R. Leroy a,∗ , T. Vanholder a , J.R. Delanghe b , G. Opsomer a , A. Van Soom a , P.E.J. Bols c , A. de Kruif a a
c
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke B-9820, Belgium b Department of Clinical Chemistry, Faculty of Medicine, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium Laboratory for Veterinary Physiology, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium Received 15 January 2003; received in revised form 20 June 2003; accepted 22 July 2003
Abstract Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to examine the biochemical composition of follicular fluid harvested from different-sized follicles and its relationship with that of blood serum in dairy cattle. Following slaughter, blood samples were collected from dairy cows (n = 30) and follicular fluid aspirated from three size classes of non-atretic follicles (<4 mm, 6–8 mm and >10 mm diameter). Samples remained independent between cows and between size classes within cows. Serum and follicular fluid samples were assayed using commercial clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and metabolites (glucose, -hydroxybutyrate (-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, -OHB and total cholesterol increased from small to large follicles and decreased for potassium, chloride, lactate, urea and triglycerides. There was a significant concentration gradient for all variables between their levels in serum and follicular fluid (P < 0.05). Significant correlations were observed for chloride (r = 0.40), glucose (r = 0.56), -OHB (r = 0.85), urea (r = 0.95) and total protein (r = 0.60) for all three follicle size classes and for triglycerides (r = 0.43), NEFA (r = 0.50) and total cholesterol (r = 0.42) for large follicles (P < 0.05). The results from the present study suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical environment that changes from small to large follicles. Furthermore, the significant correlation ∗ Corresponding author. Tel.: +32-9-264-7548; fax: +32-9-264-7597. E-mail address: [email protected] (J.L.M.R. Leroy).
0378-4320/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-4320(03)00173-8
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between the composition of serum and follicular fluid for the above-mentioned metabolites suggests that metabolic changes in serum levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells. © 2003 Elsevier B.V. All rights reserved. Keywords: Cattle ovary; Fertility; Follicular fluid composition; Metabolites; Serum levels
1. Introduction Metabolic changes in serum concentrations, caused by a negative energy balance and a high-energy and high-protein diet, occur in some high-yielding dairy cows shortly after parturition. These changes can induce pathological conditions such as hypoglycemia, ketonemia, uremia, hyperlipidemia, hypercholesterolemia, and increased levels of non-esterified fatty acids (NEFA) which may have a deleterious effect on fertility in dairy cows (Butler and Smith, 1989; Harrison et al., 1990; Butler, 1998; Opsomer, 1999; Bertoni et al., 2002). O’Callaghan and Boland (1999) suggested that the decline in fertility in high-yielding dairy cattle is mainly a problem of inferior oocyte and embryo quality, rather than being the result of a disruption in gonadotropin secretion. Since it has already been shown that changes in concentrations of gonadotropins, steroids and growth factors in follicular fluid of dairy cows were linked with alterations in oocyte quality (Wehrman et al., 1993; Izadyar et al., 1997; Driancourt and Thuel, 1998), it is not unlikely that metabolites which are present in the follicular fluid can influence oocyte quality. Moreover, several in vitro studies showed that metabolites, such as glucose, urea and -hydroxybutyrate (-OHB) may influence the competence of bovine oocytes to mature and, after fertilization, to grow to the blastocyst stage (Gomez, 1997; Hashimoto et al., 2000; Armstrong et al., 2001; De Wit et al., 2001). The follicular fluid forms the biochemical environment of the oocyte before ovulation (Edwards, 1974; Chang et al., 1976; Gosden et al., 1988; Józwik et al., 2001). It is an avascular compartment within the mammalian ovary, separated from the perifollicular stroma by the follicular wall, that constitutes a ‘blood–follicle barrier’ (Okuda et al., 1982; Bagavandoss et al., 1983). Follicular fluid is in part an exudate of serum and is in addition partially composed of locally produced substances, which are related to the metabolic activity of follicular cells (Gérard et al., 2002). This metabolic activity, together with the ‘barrier’ properties of the follicular wall, is changing significantly during the growth phase of the follicle (Edwards, 1974; Zamboni, 1974; Bagavandoss et al., 1983; Wise, 1987; Gosden et al., 1988). Therefore, a different biochemical composition of the follicular fluid in different-sized follicles can be expected. Before focusing on possible effects of metabolic changes on follicle and oocyte quality, it is necessary to determine physiological concentrations of the most common metabolites in follicular fluid from different-sized follicles and to investigate to what extent the serum and follicular fluid levels are correlated. Therefore, the aims of this study were (1) to determine the chemical composition of follicular fluid in three different follicle sizes and (2) to compare and correlate the biochemical composition of serum and of follicular fluid. Because of their importance in the metabolism of dairy cows, concentrations of glucose, -OHB, lactate, urea, total protein, triglycerides, NEFA and total cholesterol were examined. In addition,
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the basic ionic composition (sodium, potassium and chloride) of the follicular fluid and the serum was investigated. 2. Materials and methods 2.1. Animals, ovary collection and sample preparation Thirty adult dairy cows (Holstein Friesian) in good health and with normal reproductive tracts upon macroscopical examination after slaughter were used for this study. No pre-slaughter information was available for these animals. Collection of all samples was performed on two different days of the same week. Ovaries were collected immediately after slaughter and a blood sample was taken into capped disposable plastic tubes (unheparinized) during exsanguination. Both ovaries and the blood sample were identified by using the eartag number of the cow. Blood was allowed to coagulate for 20 min at 15 ◦ C and then cooled at 4 ◦ C, after which the ovaries and blood samples were transported on ice (4 ◦ C) to the laboratory. Ovaries were washed twice in cooled 0.9% NaCl (4 ◦ C) and blotted dry. Three different follicle classes, based on follicle diameter, were considered for puncture: small follicles (<4 mm), medium follicles (6–8 mm) and large follicles (>10 mm). Follicular fluid was collected by aspiration with a 26 G needle and a 1 ml syringe and pooled per follicle class within cow. For each cow and follicle class, a different needle and syringe were used. Haemorrhagic and morphologically atretic follicles, identified macroscopically according to the method of Kruip and Dieleman (1982), were not sampled. Follicular fluid (at least 0.3 ml per sample) was centrifuged (10, 000 × g, 7 min) and the supernatant was collected for analysis. The coagulated blood samples were centrifuged (1400×g, 30 min) within 1.5 h after collection and the serum was separated. Sample preparation was completed within 3 h after slaughter. Samples were snap-frozen in CO2 ice (−65 ◦ C) and stored at −20 ◦ C until biochemical assay, which took place within 3 days of ovary collection. 2.2. Biochemical analyses In each sample, the concentrations of sodium, potassium, chloride, glucose, lactate, -OHB, urea, total protein, triglycerides, NEFA and total cholesterol were measured. All analyses were performed at the Department of Clinical Chemistry, University Hospital, Ghent, Belgium. The determination of metabolite levels in follicular fluid and blood serum was done using wet chemistry techniques on two clinical chemistry autoanalysers (Modular P and Hitachi 911, Roche Diagnostics). Sodium, potassium and chloride were measured using indirect potentiometry. Measurements of glucose, lactate, urea, total protein, triglycerides and total cholesterol were performed using commercial photometric assays (Roche Diagnostics GmbH, Mannheim, Germany). Commercial kits were also used for the measurement of -OHB (Sigma Diagnostics Inc., St. Louis, USA) and NEFA (Wako Chemicals GmbH, Neuss, Germany). All measurements were carried out according to the manufacturers’ instructions. The intra- and inter-assay coefficients of variation for all analyses were below 5%.
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2.3. Statistical analyses Results are expressed as means ± S.E.M. The overall mean concentration ± S.E.M. of each metabolite and ion was calculated for follicular fluid and for blood serum in all cows. The concentrations of each factor in the follicular fluid were compared between the three follicle classes. A comparison was made for the levels in the follicular fluid of each follicle class and those of serum. Concentrations in the three different follicle classes were compared using a Linear Mixed Effects Model (S-PLUS 2000, Cambridge, USA) in which the cow is considered as a random effect. Correlation coefficients between follicular fluid and serum levels of the same parameter were calculated, and a paired samples t-test was performed to compare concentrations found in the blood serum and the follicular fluid (SPSS 10.0 for Windows, Chicago, IL, USA). A value of P < 0.05 was considered statistically significant.
3. Results An average (±S.E.M.) of 16.5 ± 1.3 small, 2.73 ± 0.3 medium and 1.33 ± 0.2 large follicles were punctured per cow. A larger number of small follicles was needed to obtain a sufficient amount of follicular fluid for analysis. The concentration of ions and metabolites in the follicular fluid from small, medium and large follicles is shown in Tables 1 and 2. Potassium, chloride, lactate, urea and triglyceride concentrations decreased significantly as follicle size increased (P < 0.05). The proportionate decrease was 61% for lactate and 43% for triglycerides. Conversely, the concentrations of glucose -OHB and total cholesterol increased as follicle size increased and the values for glucose and -OHB rose by 46 and 33%, respectively, from small to large follicles. The increase in total cholesterol was smaller but still significant (P < 0.05). The concentrations of glucose, lactate, -OHB, urea, NEFA and total cholesterol in all follicle classes varied considerably between animals.
Table 1 Average concentrations (±S.E.M.) of ions (sodium, potassium and chloride), glucose and lactate in follicular fluid of each follicle class and in serum of 30 dairy cows
Small follicles (<4 mm) Medium follicles (6–8 mm) Large follicles (>10 mm) Blood serum
Na (mM)
K (mM)
Cl (mM)
Glucose (mM)
Lactate (mM)
142.5 ± 0.34 a
10.1 ± 0.21 a∗∗∗ 105.0 ± 0.50 a∗∗ 2.01 ± 0.10 a∗∗∗
14.4 ± 0.35 a∗∗∗
142.4 ± 0.63 a
7.9 ± 0.28 b∗∗∗ 104.0 ± 0.60 b∗∗ 2.85 ± 0.16 b∗∗∗
9.4 ± 0.35 b∗∗∗
141.0 ± 1.14 a
6.0 ± 0.23 c∗∗∗ 102.9 ± 0.76 c∗∗ 3.75 ± 0.18 c∗∗∗
5.6 ± 0.37 c∗∗∗
145.0 ± 0.641,2,3,∗ 5.0 ± 0.101,2,3,∗ 102.1 ± 0.641,2,∗ 4.77 ± 0.111,2,3,∗ 5.0 ± 0.321,2,∗
a, b, c: Data with different letters within a column differ significantly between follicle classes. Superscript numbers 1, 2, 3: Concentrations in serum differ significantly from the concentrations found in small, medium and large follicles, respectively. ∗ Statistical level of significance: P < 0.05. ∗∗ Statistical level of significance: P < 0.01. ∗∗∗ Statistical level of significance: P < 0.001.
Small follicles (<4 mm) Medium follicles (6–8 mm) Large follicles (>10 mm) Blood serum
-OHB (mM)
Urea (mM)
Total protein (g/dl)
Triglycerides (mg/dl)
NEFA (mM)
Total cholesterol (mg/dl)
0.29 ± 0.02 a∗ 0.39 ± 0.03 b∗ 0.43 ± 0.03 c∗ 0.33 ± 0.022,3,∗
4.65 ± 0.35 a∗∗∗ 4.30 ± 0.34 b∗∗∗ 4.13 ± 0.34 c∗∗∗ 4.00 ± 0.291,2,∗
6.59 ± 0.10 a 6.36 ± 0.11 a 6.50 ± 0.10 a 8.19 ± 0.111,2,3,∗
21.8 ± 0.60 a∗∗∗ 16.6 ± 0.55 b∗∗∗ 12.4 ± 0.45 c∗∗∗ 17.0 ± 0.911,3,∗
0.47 ± 0.04 a 0.50 ± 0.04 a 0.44 ± 0.08 a 0.58 ± 0.083,∗
55.9 ± 3.39 a∗ 62.7 ± 2.91 b∗ 63.7 ± 3.23 c∗ 147.9 ± 9.361,2,3,∗
a, b, c: Data with different superscripts within a column differ significantly between follicle classes. Superscript numbers 1, 2, 3: Concentrations in serum differ significantly from the concentrations found in small, medium and large follicles, respectively. ∗ Statistical level of significance: P < 0.05. ∗∗∗ Statistical level of significance: P < 0.001.
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Table 2 Average concentrations (± S.E.M.) of -OHB, urea, total protein, triglycerides, NEFA and total cholesterol in follicular fluid of each follicle class and in serum of 30 dairy cows
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Table 3 Correlation coefficients (r’s) between ion and metabolite concentrations in follicular fluid and serum for each size of follicle Correlation (r)
Na
K
Cl
Glucose Lactate -OHB Urea Total Triglyceride NEFA Cholesterol protein
Serum–small 0.47 NS 0.65 0.62 follicle Serum–medium NS NS 0.74 0.48 follicle Serum–large NS NS 0.40 0.56 follicle
0.48
0.56
0.90 0.71
NS
NS
NS
NS
0.86
0.92 0.63
NS
NS
0.66
NS
0.85
0.95 0.60
0.43
0.50
0.42
Values are presented for significant correlations (P < 0.05; NS: not significant).
The ion and metabolite serum levels of the 30 cows are presented in Tables 1 and 2. The concentration of lactate, -OHB, urea, triglyceride, NEFA and total cholesterol varied considerably between animals (coefficient of variance = 35.5, 39.4, 41.5, 29.1, 75.9 and 34.6% respectively). The serum concentrations of sodium, glucose, total protein and total cholesterol were significantly higher than in small, medium and large follicles (P < 0.05). The average total protein and cholesterol concentrations found in all follicular classes were 80 and 41%, respectively, of the concentration found in serum. The concentration of glucose in follicular fluid of small follicles was less than half of the level found in serum but only 8
urea concentration in large follicles (mM)
7
6 5 4
3 2
1 0 1
2
3
4
5
6
7
urea concentration in serum (mM) Fig. 1. Relationship between the urea concentration in serum and in follicular fluid of large follicles in 30 dairy cows (r = 0.953, P < 0.05).
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21% lower in large follicles. Potassium concentrations in serum were significantly lower than the levels in all follicle classes and only half of the concentration found in small follicles (P < 0.05). Chloride, lactate and urea levels in small- and medium-sized follicles were significantly higher than the serum levels, but the values for large follicles were similar to those in serum (P < 0.05). -OHB was the only factor with a significantly lower concentration in blood serum compared to the levels in medium and large follicles (P < 0.05). The serum concentration of triglycerides was significantly lower than the level measured in small follicles, (P < 0.05), similar in value to that in medium follicles but higher than in large follicles (P < 0.05). NEFA concentrations were lower in all follicle sizes than in serum, but the difference was significant only for large follicles (P < 0.05). The correlation coefficients between serum and follicular fluid of each follicle class were calculated, and significant (P < 0.05) correlation coefficients (r’s) are presented in Table 3. High correlations between serum levels and levels in follicular fluid of all three follicular classes were found for chloride, glucose, -OHB, urea and total protein. The highest correlation observed was for urea (Fig. 1). The coefficient for -OHB was also high in medium and large follicles. There was no significant correlation between serum and follicular fluid potassium levels for any follicle class. A significant correlation was observed between concentrations in the follicular fluid of large follicles and in serum for all variates except sodium, potassium and lactate.
4. Discussion In the present study, follicular fluid was sampled separately for each cow unlike in previous studies (Chang et al., 1976; Cabrera et al., 1985; Wise, 1987; Hammon et al., 2000), where follicular fluid samples from different cows were pooled for technical reasons. Obviously atretic follicles were excluded from aspiration (Chang et al., 1976; Homa and Brown, 1992), but because up to 85% of all bovine antral follicles show signs of atresia, it is possible that a proportion of follicles sampled in all follicle classes were atretic (Kruip and Dieleman, 1982). The sodium, chloride and potassium concentration in the follicular fluid were similar to those given in other studies (Chang et al., 1976; Wise, 1987; Collins et al., 1997). The concentration gradient for potassium between serum and follicular fluid suggests an active inward transport of the cation (Gosden et al., 1988). Moreover, no correlation with serum was found, indicating that potassium levels in follicular fluid may also be the result of local metabolism. Glucose plays an important role in ovarian metabolism since it is the major energy source for the bovine, mouse and human ovary, possibly metabolized by the ovary through anaerobic pathways, leading to lactate formation (Leese and Lenton, 1990; Boland et al., 1994; Rabiee et al., 1997b, 1999). We found that glucose and lactate concentrations in follicular fluid were lower, and respectively higher than those measured in serum. Our data also show that the glucose concentration increases and lactate levels decrease when the follicle diameter increases, which confirms the results of Landau et al. (2000) for dairy cows and Chang et al. (1976) for sows. This could mean that glucose metabolism is less intensive in large follicles compared with small ones, resulting in a lower consumption of glucose from follicular fluid and in a reduced secretion of lactate into the follicular fluid. An
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increasing amount of follicular fluid is a second explanation for the increase in glucose and the decrease in lactate, since in large follicles a relatively smaller number of granulosa cells consumes glucose from and secretes lactate into a relatively larger amount of follicular fluid (McNatty et al., 1978; Gosden et al., 1988). A further reason for this observation could be the increased permeability of the blood–follicle barrier during follicular growth (Edwards, 1974; Zamboni, 1974; Okuda et al., 1982; Bagavandoss et al., 1983). Consequently, an equilibrium between the vascular compartment and follicular fluid can be achieved more easily in large follicles. Leese and Lenton (1990) concluded that the glucose and lactate concentrations in follicular fluid in women is a result of both glycolysis taking place in the mural granulosa cells and influx of the same molecules from the plasma into the fluid. This is supported by our data, which show good correlations between blood serum and follicular fluid for glucose at each follicular size. Hence, these correlation coefficients suggest that hypoglycemia may reduce the glucose content in follicular fluid, but this needs to be confirmed by further investigation in hypoglycemic dairy cows. When interpreting these kind of data for potassium, glucose and lactate, it is important to consider postmortem changes which can induce increased potassium concentrations by leakage from damaged cells and turnover of glucose to lactate by anaerobic glycolysis (Chang et al., 1976; Gosden et al., 1988). In a preliminary study, no other metabolite concentrations changed significantly during transport to the laboratory (Leroy et al., unpublished). The strong correlation between -OHB levels in follicular fluid of all three classes of follicles and serum suggest that elevated levels in the serum of dairy cows (ketonemia) may cause similar changes in the follicular fluid. Further studies with ketonemic cows are required to confirm this conclusion. The significant increase of -OHB from small to large follicles is possibly caused by a local secretion of this ketone body by the follicle cells. This needs, however, further investigation. Rabiee et al. (1999) showed that -OHB can be used or converted by the bovine and ovine ovary (Rabiee et al., 1997a,b, 1999). The observation that follicular levels of urea were different between all follicle classes was not expected. In small- and medium-sized follicles, the concentration of urea was significantly higher than the serum concentrations, possibly caused by an active inward transport or a local urea production by the follicle cells. As Collins et al. (1997) found in mares, we found a very high correlation for urea between follicular fluid and blood serum. Reports about the effect of elevated urea levels on fertility are contradictory, although all authors agree that the possible adverse effect of diet-induced elevated urea levels must act at the level of the oocyte (Sinclair et al., 2000; Dawuda et al., 2002). The high correlation between follicular fluid and serum suggests that elevated serum urea levels of dairy cows may be reflected in the follicular fluid and hence, may influence oocyte quality. This requires further investigation. The total protein content of the follicular fluid did not differ between follicle classes, and was about 75% of that present in serum. The high correlation between total protein content in follicular fluid and in serum suggests that a substantial part of the protein content in follicular fluid originates from serum (Edwards, 1974; Wise, 1987). The amount of triglycerides decreased from small to large follicles. Triglyceride levels in small follicles were significantly higher than in serum and significantly lower in large follicles. There was a significant correlation between the follicular fluid and the serum levels of triglycerides only in large follicles. These data favour the idea that follicular triglyceride
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levels are mainly a result of local metabolic processes. A relatively stable concentration of triglycerides is maintained in the bovine ovarian follicle, regardless of increases in serum due to physiological status or diet (Wehrman et al., 1991). Triglycerides probably do not pass through the follicular membrane since they are transported primarily by the very low-density lipoprotein fraction (VLDL), which is too large to pass through this barrier (Grummer and Carroll, 1988). In follicular fluid, triglycerides may serve as an alternative energy source since cells cultured in vitro can absorb and consume triglycerides out of the medium. Also, oocytes and embryos show lipid accumulation when cultured in triglyceride containing media (Kim et al., 2001, Abe et al., 2002). This also applies for NEFA (Abe et al., 2002). NEFA are transported in the blood by means of albumin, and this complex can easily penetrate the follicular wall. NEFA concentrations did not differ between the different follicle classes and tended to be higher in serum. A significant correlation with serum levels of NEFA was observed only for large follicles. Total cholesterol in follicular fluid was about 42% of the concentration found in blood serum and there was a significant increase of the total cholesterol content from small to large follicles. Cholesterol, present in follicular fluid, is bound to the high-density lipoprotein fraction (HDL) because the only other cholesterol-containing lipoprotein fraction, the low-density lipoprotein fraction (LDL), is too large to pass the blood–follicle barrier (Puppione, 1977; Grummer and Carroll, 1988; Wehrman et al., 1991; Bauchart, 1993). The higher total cholesterol concentration in large follicles can be explained by the increased permeability of the follicular wall in that follicle class, permitting the entrance of the larger HDL fraction (Bagavandoss et al., 1983; Wehrman et al., 1991). A significant correlation between follicular fluid and serum levels of cholesterol was noticed in medium and large follicles.
5. Conclusion In conclusion, we observed an increase in the concentrations of glucose, -OHB and total cholesterol and a decrease in the concentrations of potassium, chloride, lactate, urea and triglycerides in the follicular fluid from small to large follicles. Although we have not evaluated changes in the biochemical composition of follicular fluid from one follicle during its growth, our data, however, suggest that what we found in the different-sized follicles reflects what happens during the follicular growth. Our findings suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a changing biochemical environment from small to large follicles and that this environment is correlated with serum levels of the ions and metabolites studied here. Further research should concentrate on changes in these metabolites in the follicular fluid of high-producing dairy cows in vivo during the first weeks of lactation and their effect on oocyte quality.
Acknowledgements The authors thank Dr. M. Berth for his excellent scientific and technical support, Dr. K. Moerloose for the critical reading of the manuscript, and Drs. J. Dewulf and S. De Vliegher
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Homa, S.T., Brown, C.A., 1992. Changes in linoleic acid during follicular development and inhibition of spontaneous breakdown of germinal vesicles in cumulus-free bovine oocytes. J. Reprod. Fertil. 94, 153–160. Izadyar, F., Van Tol, H.T., Colenbrander, B., Bevers, M.M., 1997. Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through cumulus cells and not mediated by IGF-I. Mol. Reprod. Dev. 47, 175–180. Józwik, M., Józwik, M., Wolczynski, S., Józwik, M., Szamatowicz, M., 2001. Ammonia concentration in human preovulatoy ovarian follicles. Eur. J. Obstet. Gynecol. Reprod. Biol. 94, 256–260. Kim, J.Y., Kinoshita, M., Ohnishi, M., Fukui, Y., 2001. Lipid and fatty acid analysis of fresh and frozen-thawed immature and in vitro matured bovine oocytes. Reproduction 122, 131–138. Kruip, T.A.M., Dieleman, S.J., 1982. Macroscopic classification of bovine follicles and its validation by micromorpholigical and steroid biochemical procedures. Reprod. Nutr. Dev. 22, 465–473. Landau, S., Braw-Tal, R., Kaim, M., Bor, A., Bruckental, I., 2000. Preovulatory follicular status and diet affect the insulin and glucose content of follicles in high-yielding dairy cows. Anim. Reprod. Sci. 64, 181–197. Leese, H.J., Lenton, E.A., 1990. Glucose and lactate in human follicular fluid: concentrations and interrelationships. Hum. Reprod. 5, 915–919. McNatty, K.P., Smith, D.M., Makris, A., Osathanondh, R., Ryan, K.J., 1978. The microenvironment of the human antral follicle: interrelationships among the steroid levels in the antral fluid, the population of granulosa cells, and the status of the oocyte in vivo and in vitro. J. Clin. Endocrinol. Metab. 49, 851–860. O’Callaghan, D., Boland, M.P., 1999. Nutritional effects on ovulation. Anim. Sci. 68, 299–314. Okuda, Y., Okamura, H., Kanzaki, H., Takenaka, A., Morimoto, K., Nishimura, T., 1982. An ultrastructural study of capillary permeability of rabbit ovarian follicles using horseradish peroxidase as a tracer. Nippon Sanka Fujinka Gakkai Zasshi 34, 181–186. Opsomer, G., 1999. Anoestrus Postpartum in High Yielding Dairy Cows: A Field Study, Ph.D. Thesis, Faculty of Veterinary Medicine, Ghent University. Puppione, D.L., 1977. Implications of unique features of blood lipid transport in the lactating cow. J. Dairy Sci. 61, 651–659. Rabiee, A.R., Lean, I.J., Gooden, J.M., Miller, B.G., 1997a. Short-term studies of ovarian metabolism in the ewe. Anim. Reprod. Sci. 47, 43–58. Rabiee, A.R., Lean, I.J., Gooden, J.M., Miller, B.G., Scaramuzzi, R.J., 1997b. An evaluation of transovarian uptake of metabolites using arterio-venous differences methods in dairy cattle. Anim. Reprod. Sci. 48, 9–25. Rabiee, A.R., Lean, I.J., Gooden, J.M., Miller, B.G., 1999. Relationships among metabolites influencing ovarian function in the dairy cow. J. Dairy Sci. 82, 39–44. Sinclair, K.D., Kuran, M., Gebbie, F.E., Webb, R., McEvoy, T.G., 2000. Nitrogen metabolism and fertility in cattle: II. Development of oocytes recovered from heifers offered diets differing in their rate of nitrogen release in the rumen. J. Anim. Sci. 78, 2670–2680. Wehrman, M.E., Welsh, T.H., Williams, G.L., 1991. Diet-induced hyperlipidemia in cattle modifies the intrafollicular cholesterol environment, modulates ovarian follicular dynamics, and hastens the onset of postpartum luteal activity. Biol. Reprod. 45, 514–522. Wehrman, M.E., Roberson, M.S., Cupp, A.S., Kojima, F.N., Stumpf, T.T., Werth, L.A., Wolfe, M.W., Kittok, R.J., Kinder, J.E., 1993. Increasing exogenous progesterone during synchronization of estrus decreases endogenous 17-estradiol and increases conception in cows. Biol. Reprod. 49, 214–220. Wise, T., 1987. Biochemical analysis of bovine follicular fluid: albumin, total protein, lysosomal enzymes, ions, steroids and ascorbic acid content in relation to follicular size, rank, atresia classification and day of estrous cycle. J. Anim. Sci. 64, 1153–1169. Zamboni, L., 1974. Fine morphology of the follicle cell–oocyte association. Biol. Reprod. 10, 125–149.
Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows J.L.M.R. Leroy a,∗ , T. Vanholder a , J.R. Delanghe b , G. Opsomer a , A. Van Soom a , P.E.J. Bols c , A. de Kruif a a
c
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke B-9820, Belgium b Department of Clinical Chemistry, Faculty of Medicine, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium Laboratory for Veterinary Physiology, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium Received 15 January 2003; received in revised form 20 June 2003; accepted 22 July 2003
Abstract Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to examine the biochemical composition of follicular fluid harvested from different-sized follicles and its relationship with that of blood serum in dairy cattle. Following slaughter, blood samples were collected from dairy cows (n = 30) and follicular fluid aspirated from three size classes of non-atretic follicles (<4 mm, 6–8 mm and >10 mm diameter). Samples remained independent between cows and between size classes within cows. Serum and follicular fluid samples were assayed using commercial clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and metabolites (glucose, -hydroxybutyrate (-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, -OHB and total cholesterol increased from small to large follicles and decreased for potassium, chloride, lactate, urea and triglycerides. There was a significant concentration gradient for all variables between their levels in serum and follicular fluid (P < 0.05). Significant correlations were observed for chloride (r = 0.40), glucose (r = 0.56), -OHB (r = 0.85), urea (r = 0.95) and total protein (r = 0.60) for all three follicle size classes and for triglycerides (r = 0.43), NEFA (r = 0.50) and total cholesterol (r = 0.42) for large follicles (P < 0.05). The results from the present study suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical environment that changes from small to large follicles. Furthermore, the significant correlation ∗ Corresponding author. Tel.: +32-9-264-7548; fax: +32-9-264-7597. E-mail address: [email protected] (J.L.M.R. Leroy).
0378-4320/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-4320(03)00173-8
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between the composition of serum and follicular fluid for the above-mentioned metabolites suggests that metabolic changes in serum levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells. © 2003 Elsevier B.V. All rights reserved. Keywords: Cattle ovary; Fertility; Follicular fluid composition; Metabolites; Serum levels
1. Introduction Metabolic changes in serum concentrations, caused by a negative energy balance and a high-energy and high-protein diet, occur in some high-yielding dairy cows shortly after parturition. These changes can induce pathological conditions such as hypoglycemia, ketonemia, uremia, hyperlipidemia, hypercholesterolemia, and increased levels of non-esterified fatty acids (NEFA) which may have a deleterious effect on fertility in dairy cows (Butler and Smith, 1989; Harrison et al., 1990; Butler, 1998; Opsomer, 1999; Bertoni et al., 2002). O’Callaghan and Boland (1999) suggested that the decline in fertility in high-yielding dairy cattle is mainly a problem of inferior oocyte and embryo quality, rather than being the result of a disruption in gonadotropin secretion. Since it has already been shown that changes in concentrations of gonadotropins, steroids and growth factors in follicular fluid of dairy cows were linked with alterations in oocyte quality (Wehrman et al., 1993; Izadyar et al., 1997; Driancourt and Thuel, 1998), it is not unlikely that metabolites which are present in the follicular fluid can influence oocyte quality. Moreover, several in vitro studies showed that metabolites, such as glucose, urea and -hydroxybutyrate (-OHB) may influence the competence of bovine oocytes to mature and, after fertilization, to grow to the blastocyst stage (Gomez, 1997; Hashimoto et al., 2000; Armstrong et al., 2001; De Wit et al., 2001). The follicular fluid forms the biochemical environment of the oocyte before ovulation (Edwards, 1974; Chang et al., 1976; Gosden et al., 1988; Józwik et al., 2001). It is an avascular compartment within the mammalian ovary, separated from the perifollicular stroma by the follicular wall, that constitutes a ‘blood–follicle barrier’ (Okuda et al., 1982; Bagavandoss et al., 1983). Follicular fluid is in part an exudate of serum and is in addition partially composed of locally produced substances, which are related to the metabolic activity of follicular cells (Gérard et al., 2002). This metabolic activity, together with the ‘barrier’ properties of the follicular wall, is changing significantly during the growth phase of the follicle (Edwards, 1974; Zamboni, 1974; Bagavandoss et al., 1983; Wise, 1987; Gosden et al., 1988). Therefore, a different biochemical composition of the follicular fluid in different-sized follicles can be expected. Before focusing on possible effects of metabolic changes on follicle and oocyte quality, it is necessary to determine physiological concentrations of the most common metabolites in follicular fluid from different-sized follicles and to investigate to what extent the serum and follicular fluid levels are correlated. Therefore, the aims of this study were (1) to determine the chemical composition of follicular fluid in three different follicle sizes and (2) to compare and correlate the biochemical composition of serum and of follicular fluid. Because of their importance in the metabolism of dairy cows, concentrations of glucose, -OHB, lactate, urea, total protein, triglycerides, NEFA and total cholesterol were examined. In addition,
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the basic ionic composition (sodium, potassium and chloride) of the follicular fluid and the serum was investigated. 2. Materials and methods 2.1. Animals, ovary collection and sample preparation Thirty adult dairy cows (Holstein Friesian) in good health and with normal reproductive tracts upon macroscopical examination after slaughter were used for this study. No pre-slaughter information was available for these animals. Collection of all samples was performed on two different days of the same week. Ovaries were collected immediately after slaughter and a blood sample was taken into capped disposable plastic tubes (unheparinized) during exsanguination. Both ovaries and the blood sample were identified by using the eartag number of the cow. Blood was allowed to coagulate for 20 min at 15 ◦ C and then cooled at 4 ◦ C, after which the ovaries and blood samples were transported on ice (4 ◦ C) to the laboratory. Ovaries were washed twice in cooled 0.9% NaCl (4 ◦ C) and blotted dry. Three different follicle classes, based on follicle diameter, were considered for puncture: small follicles (<4 mm), medium follicles (6–8 mm) and large follicles (>10 mm). Follicular fluid was collected by aspiration with a 26 G needle and a 1 ml syringe and pooled per follicle class within cow. For each cow and follicle class, a different needle and syringe were used. Haemorrhagic and morphologically atretic follicles, identified macroscopically according to the method of Kruip and Dieleman (1982), were not sampled. Follicular fluid (at least 0.3 ml per sample) was centrifuged (10, 000 × g, 7 min) and the supernatant was collected for analysis. The coagulated blood samples were centrifuged (1400×g, 30 min) within 1.5 h after collection and the serum was separated. Sample preparation was completed within 3 h after slaughter. Samples were snap-frozen in CO2 ice (−65 ◦ C) and stored at −20 ◦ C until biochemical assay, which took place within 3 days of ovary collection. 2.2. Biochemical analyses In each sample, the concentrations of sodium, potassium, chloride, glucose, lactate, -OHB, urea, total protein, triglycerides, NEFA and total cholesterol were measured. All analyses were performed at the Department of Clinical Chemistry, University Hospital, Ghent, Belgium. The determination of metabolite levels in follicular fluid and blood serum was done using wet chemistry techniques on two clinical chemistry autoanalysers (Modular P and Hitachi 911, Roche Diagnostics). Sodium, potassium and chloride were measured using indirect potentiometry. Measurements of glucose, lactate, urea, total protein, triglycerides and total cholesterol were performed using commercial photometric assays (Roche Diagnostics GmbH, Mannheim, Germany). Commercial kits were also used for the measurement of -OHB (Sigma Diagnostics Inc., St. Louis, USA) and NEFA (Wako Chemicals GmbH, Neuss, Germany). All measurements were carried out according to the manufacturers’ instructions. The intra- and inter-assay coefficients of variation for all analyses were below 5%.
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2.3. Statistical analyses Results are expressed as means ± S.E.M. The overall mean concentration ± S.E.M. of each metabolite and ion was calculated for follicular fluid and for blood serum in all cows. The concentrations of each factor in the follicular fluid were compared between the three follicle classes. A comparison was made for the levels in the follicular fluid of each follicle class and those of serum. Concentrations in the three different follicle classes were compared using a Linear Mixed Effects Model (S-PLUS 2000, Cambridge, USA) in which the cow is considered as a random effect. Correlation coefficients between follicular fluid and serum levels of the same parameter were calculated, and a paired samples t-test was performed to compare concentrations found in the blood serum and the follicular fluid (SPSS 10.0 for Windows, Chicago, IL, USA). A value of P < 0.05 was considered statistically significant.
3. Results An average (±S.E.M.) of 16.5 ± 1.3 small, 2.73 ± 0.3 medium and 1.33 ± 0.2 large follicles were punctured per cow. A larger number of small follicles was needed to obtain a sufficient amount of follicular fluid for analysis. The concentration of ions and metabolites in the follicular fluid from small, medium and large follicles is shown in Tables 1 and 2. Potassium, chloride, lactate, urea and triglyceride concentrations decreased significantly as follicle size increased (P < 0.05). The proportionate decrease was 61% for lactate and 43% for triglycerides. Conversely, the concentrations of glucose -OHB and total cholesterol increased as follicle size increased and the values for glucose and -OHB rose by 46 and 33%, respectively, from small to large follicles. The increase in total cholesterol was smaller but still significant (P < 0.05). The concentrations of glucose, lactate, -OHB, urea, NEFA and total cholesterol in all follicle classes varied considerably between animals.
Table 1 Average concentrations (±S.E.M.) of ions (sodium, potassium and chloride), glucose and lactate in follicular fluid of each follicle class and in serum of 30 dairy cows
Small follicles (<4 mm) Medium follicles (6–8 mm) Large follicles (>10 mm) Blood serum
Na (mM)
K (mM)
Cl (mM)
Glucose (mM)
Lactate (mM)
142.5 ± 0.34 a
10.1 ± 0.21 a∗∗∗ 105.0 ± 0.50 a∗∗ 2.01 ± 0.10 a∗∗∗
14.4 ± 0.35 a∗∗∗
142.4 ± 0.63 a
7.9 ± 0.28 b∗∗∗ 104.0 ± 0.60 b∗∗ 2.85 ± 0.16 b∗∗∗
9.4 ± 0.35 b∗∗∗
141.0 ± 1.14 a
6.0 ± 0.23 c∗∗∗ 102.9 ± 0.76 c∗∗ 3.75 ± 0.18 c∗∗∗
5.6 ± 0.37 c∗∗∗
145.0 ± 0.641,2,3,∗ 5.0 ± 0.101,2,3,∗ 102.1 ± 0.641,2,∗ 4.77 ± 0.111,2,3,∗ 5.0 ± 0.321,2,∗
a, b, c: Data with different letters within a column differ significantly between follicle classes. Superscript numbers 1, 2, 3: Concentrations in serum differ significantly from the concentrations found in small, medium and large follicles, respectively. ∗ Statistical level of significance: P < 0.05. ∗∗ Statistical level of significance: P < 0.01. ∗∗∗ Statistical level of significance: P < 0.001.
Small follicles (<4 mm) Medium follicles (6–8 mm) Large follicles (>10 mm) Blood serum
-OHB (mM)
Urea (mM)
Total protein (g/dl)
Triglycerides (mg/dl)
NEFA (mM)
Total cholesterol (mg/dl)
0.29 ± 0.02 a∗ 0.39 ± 0.03 b∗ 0.43 ± 0.03 c∗ 0.33 ± 0.022,3,∗
4.65 ± 0.35 a∗∗∗ 4.30 ± 0.34 b∗∗∗ 4.13 ± 0.34 c∗∗∗ 4.00 ± 0.291,2,∗
6.59 ± 0.10 a 6.36 ± 0.11 a 6.50 ± 0.10 a 8.19 ± 0.111,2,3,∗
21.8 ± 0.60 a∗∗∗ 16.6 ± 0.55 b∗∗∗ 12.4 ± 0.45 c∗∗∗ 17.0 ± 0.911,3,∗
0.47 ± 0.04 a 0.50 ± 0.04 a 0.44 ± 0.08 a 0.58 ± 0.083,∗
55.9 ± 3.39 a∗ 62.7 ± 2.91 b∗ 63.7 ± 3.23 c∗ 147.9 ± 9.361,2,3,∗
a, b, c: Data with different superscripts within a column differ significantly between follicle classes. Superscript numbers 1, 2, 3: Concentrations in serum differ significantly from the concentrations found in small, medium and large follicles, respectively. ∗ Statistical level of significance: P < 0.05. ∗∗∗ Statistical level of significance: P < 0.001.
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Table 2 Average concentrations (± S.E.M.) of -OHB, urea, total protein, triglycerides, NEFA and total cholesterol in follicular fluid of each follicle class and in serum of 30 dairy cows
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Table 3 Correlation coefficients (r’s) between ion and metabolite concentrations in follicular fluid and serum for each size of follicle Correlation (r)
Na
K
Cl
Glucose Lactate -OHB Urea Total Triglyceride NEFA Cholesterol protein
Serum–small 0.47 NS 0.65 0.62 follicle Serum–medium NS NS 0.74 0.48 follicle Serum–large NS NS 0.40 0.56 follicle
0.48
0.56
0.90 0.71
NS
NS
NS
NS
0.86
0.92 0.63
NS
NS
0.66
NS
0.85
0.95 0.60
0.43
0.50
0.42
Values are presented for significant correlations (P < 0.05; NS: not significant).
The ion and metabolite serum levels of the 30 cows are presented in Tables 1 and 2. The concentration of lactate, -OHB, urea, triglyceride, NEFA and total cholesterol varied considerably between animals (coefficient of variance = 35.5, 39.4, 41.5, 29.1, 75.9 and 34.6% respectively). The serum concentrations of sodium, glucose, total protein and total cholesterol were significantly higher than in small, medium and large follicles (P < 0.05). The average total protein and cholesterol concentrations found in all follicular classes were 80 and 41%, respectively, of the concentration found in serum. The concentration of glucose in follicular fluid of small follicles was less than half of the level found in serum but only 8
urea concentration in large follicles (mM)
7
6 5 4
3 2
1 0 1
2
3
4
5
6
7
urea concentration in serum (mM) Fig. 1. Relationship between the urea concentration in serum and in follicular fluid of large follicles in 30 dairy cows (r = 0.953, P < 0.05).
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21% lower in large follicles. Potassium concentrations in serum were significantly lower than the levels in all follicle classes and only half of the concentration found in small follicles (P < 0.05). Chloride, lactate and urea levels in small- and medium-sized follicles were significantly higher than the serum levels, but the values for large follicles were similar to those in serum (P < 0.05). -OHB was the only factor with a significantly lower concentration in blood serum compared to the levels in medium and large follicles (P < 0.05). The serum concentration of triglycerides was significantly lower than the level measured in small follicles, (P < 0.05), similar in value to that in medium follicles but higher than in large follicles (P < 0.05). NEFA concentrations were lower in all follicle sizes than in serum, but the difference was significant only for large follicles (P < 0.05). The correlation coefficients between serum and follicular fluid of each follicle class were calculated, and significant (P < 0.05) correlation coefficients (r’s) are presented in Table 3. High correlations between serum levels and levels in follicular fluid of all three follicular classes were found for chloride, glucose, -OHB, urea and total protein. The highest correlation observed was for urea (Fig. 1). The coefficient for -OHB was also high in medium and large follicles. There was no significant correlation between serum and follicular fluid potassium levels for any follicle class. A significant correlation was observed between concentrations in the follicular fluid of large follicles and in serum for all variates except sodium, potassium and lactate.
4. Discussion In the present study, follicular fluid was sampled separately for each cow unlike in previous studies (Chang et al., 1976; Cabrera et al., 1985; Wise, 1987; Hammon et al., 2000), where follicular fluid samples from different cows were pooled for technical reasons. Obviously atretic follicles were excluded from aspiration (Chang et al., 1976; Homa and Brown, 1992), but because up to 85% of all bovine antral follicles show signs of atresia, it is possible that a proportion of follicles sampled in all follicle classes were atretic (Kruip and Dieleman, 1982). The sodium, chloride and potassium concentration in the follicular fluid were similar to those given in other studies (Chang et al., 1976; Wise, 1987; Collins et al., 1997). The concentration gradient for potassium between serum and follicular fluid suggests an active inward transport of the cation (Gosden et al., 1988). Moreover, no correlation with serum was found, indicating that potassium levels in follicular fluid may also be the result of local metabolism. Glucose plays an important role in ovarian metabolism since it is the major energy source for the bovine, mouse and human ovary, possibly metabolized by the ovary through anaerobic pathways, leading to lactate formation (Leese and Lenton, 1990; Boland et al., 1994; Rabiee et al., 1997b, 1999). We found that glucose and lactate concentrations in follicular fluid were lower, and respectively higher than those measured in serum. Our data also show that the glucose concentration increases and lactate levels decrease when the follicle diameter increases, which confirms the results of Landau et al. (2000) for dairy cows and Chang et al. (1976) for sows. This could mean that glucose metabolism is less intensive in large follicles compared with small ones, resulting in a lower consumption of glucose from follicular fluid and in a reduced secretion of lactate into the follicular fluid. An
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increasing amount of follicular fluid is a second explanation for the increase in glucose and the decrease in lactate, since in large follicles a relatively smaller number of granulosa cells consumes glucose from and secretes lactate into a relatively larger amount of follicular fluid (McNatty et al., 1978; Gosden et al., 1988). A further reason for this observation could be the increased permeability of the blood–follicle barrier during follicular growth (Edwards, 1974; Zamboni, 1974; Okuda et al., 1982; Bagavandoss et al., 1983). Consequently, an equilibrium between the vascular compartment and follicular fluid can be achieved more easily in large follicles. Leese and Lenton (1990) concluded that the glucose and lactate concentrations in follicular fluid in women is a result of both glycolysis taking place in the mural granulosa cells and influx of the same molecules from the plasma into the fluid. This is supported by our data, which show good correlations between blood serum and follicular fluid for glucose at each follicular size. Hence, these correlation coefficients suggest that hypoglycemia may reduce the glucose content in follicular fluid, but this needs to be confirmed by further investigation in hypoglycemic dairy cows. When interpreting these kind of data for potassium, glucose and lactate, it is important to consider postmortem changes which can induce increased potassium concentrations by leakage from damaged cells and turnover of glucose to lactate by anaerobic glycolysis (Chang et al., 1976; Gosden et al., 1988). In a preliminary study, no other metabolite concentrations changed significantly during transport to the laboratory (Leroy et al., unpublished). The strong correlation between -OHB levels in follicular fluid of all three classes of follicles and serum suggest that elevated levels in the serum of dairy cows (ketonemia) may cause similar changes in the follicular fluid. Further studies with ketonemic cows are required to confirm this conclusion. The significant increase of -OHB from small to large follicles is possibly caused by a local secretion of this ketone body by the follicle cells. This needs, however, further investigation. Rabiee et al. (1999) showed that -OHB can be used or converted by the bovine and ovine ovary (Rabiee et al., 1997a,b, 1999). The observation that follicular levels of urea were different between all follicle classes was not expected. In small- and medium-sized follicles, the concentration of urea was significantly higher than the serum concentrations, possibly caused by an active inward transport or a local urea production by the follicle cells. As Collins et al. (1997) found in mares, we found a very high correlation for urea between follicular fluid and blood serum. Reports about the effect of elevated urea levels on fertility are contradictory, although all authors agree that the possible adverse effect of diet-induced elevated urea levels must act at the level of the oocyte (Sinclair et al., 2000; Dawuda et al., 2002). The high correlation between follicular fluid and serum suggests that elevated serum urea levels of dairy cows may be reflected in the follicular fluid and hence, may influence oocyte quality. This requires further investigation. The total protein content of the follicular fluid did not differ between follicle classes, and was about 75% of that present in serum. The high correlation between total protein content in follicular fluid and in serum suggests that a substantial part of the protein content in follicular fluid originates from serum (Edwards, 1974; Wise, 1987). The amount of triglycerides decreased from small to large follicles. Triglyceride levels in small follicles were significantly higher than in serum and significantly lower in large follicles. There was a significant correlation between the follicular fluid and the serum levels of triglycerides only in large follicles. These data favour the idea that follicular triglyceride
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levels are mainly a result of local metabolic processes. A relatively stable concentration of triglycerides is maintained in the bovine ovarian follicle, regardless of increases in serum due to physiological status or diet (Wehrman et al., 1991). Triglycerides probably do not pass through the follicular membrane since they are transported primarily by the very low-density lipoprotein fraction (VLDL), which is too large to pass through this barrier (Grummer and Carroll, 1988). In follicular fluid, triglycerides may serve as an alternative energy source since cells cultured in vitro can absorb and consume triglycerides out of the medium. Also, oocytes and embryos show lipid accumulation when cultured in triglyceride containing media (Kim et al., 2001, Abe et al., 2002). This also applies for NEFA (Abe et al., 2002). NEFA are transported in the blood by means of albumin, and this complex can easily penetrate the follicular wall. NEFA concentrations did not differ between the different follicle classes and tended to be higher in serum. A significant correlation with serum levels of NEFA was observed only for large follicles. Total cholesterol in follicular fluid was about 42% of the concentration found in blood serum and there was a significant increase of the total cholesterol content from small to large follicles. Cholesterol, present in follicular fluid, is bound to the high-density lipoprotein fraction (HDL) because the only other cholesterol-containing lipoprotein fraction, the low-density lipoprotein fraction (LDL), is too large to pass the blood–follicle barrier (Puppione, 1977; Grummer and Carroll, 1988; Wehrman et al., 1991; Bauchart, 1993). The higher total cholesterol concentration in large follicles can be explained by the increased permeability of the follicular wall in that follicle class, permitting the entrance of the larger HDL fraction (Bagavandoss et al., 1983; Wehrman et al., 1991). A significant correlation between follicular fluid and serum levels of cholesterol was noticed in medium and large follicles.
5. Conclusion In conclusion, we observed an increase in the concentrations of glucose, -OHB and total cholesterol and a decrease in the concentrations of potassium, chloride, lactate, urea and triglycerides in the follicular fluid from small to large follicles. Although we have not evaluated changes in the biochemical composition of follicular fluid from one follicle during its growth, our data, however, suggest that what we found in the different-sized follicles reflects what happens during the follicular growth. Our findings suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a changing biochemical environment from small to large follicles and that this environment is correlated with serum levels of the ions and metabolites studied here. Further research should concentrate on changes in these metabolites in the follicular fluid of high-producing dairy cows in vivo during the first weeks of lactation and their effect on oocyte quality.
Acknowledgements The authors thank Dr. M. Berth for his excellent scientific and technical support, Dr. K. Moerloose for the critical reading of the manuscript, and Drs. J. Dewulf and S. De Vliegher
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