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Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles
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Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles S.M. El-Bahr a,b,∗ , I.M. Ghoneim c , M.M. Waheed c a Department of Physiology, Biochemistry and Pharmacology (Biochemistry), College of Veterinary Medicine and Animal Resources, King Faisal University, Saudi Arabia b Department of Biochemistry, Faculty of Veterinary Medicine, Alexandria University, Egypt c Department of Clinical Studies, College of Veterinary Medicine and Animal Resources, King Faisal University, Saudi Arabia
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Article history: Received 25 February 2015 Received in revised form 21 May 2015 Accepted 3 June 2015 Available online xxx Keywords: Camels Different sized follicles Follicular fluid Serum Hormones Biochemistry
a b s t r a c t The current study aimed to compare some biochemical and hormonal constituents in follicular fluids and serum of female dromedary camels with different sized ovarian follicles. Therefore, follicular fluids from follicles sized 1.1–1.5 cm (n = 10), 1.6–2.1 cm (n = 10) and 2.2–2.5 cm (n = 10) and sera were harvested from 20 female camels. The concentrations of ascorbic acid, glucose, cholesterol and activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) were not changed significantly neither in follicular fluids of all follicle sizes nor in sera of female camels with different sized follicles. The concentrations of estradiol-17 (E2) in the follicular fluid of follicles sized 2.2–2.5 cm were significantly lower (P < 0.01) than its corresponding value in follicular fluid of other follicle sizes. The concentrations of progesterone (P4 ), tri-iodothyronine (T3 ), thyroxin (T4 ), cortisol and insulin-like growth factor-1 (IGF-1) remained comparable in follicular fluids of all examined different sized follicles. The concentrations of E2, P4 , T3 , T4 , cortisol and IGF-1 were similar in the serum of camels with different sized follicles. Interestingly, mean concentrations of P4 and IGF-1 in follicular fluids were higher than their corresponding values in sera of camels with different sized follicles and the mean concentrations of glucose, cholesterol, ALP and cortisol in sera were higher than their corresponding values in follicular fluids of the examined camels. With the exception of E2, there were no significant differences in biochemical and hormonal constituents between follicular fluids from different sized follicles. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The dromedary camel (Camelus dromedarius) is a seasonal breeder with a relatively short breeding season
∗ Corresponding author at: Department of Physiology, Biochemistry and Pharmacology (Biochemistry), College of Veterinary Medicine and Animal Resources, King Faisal University, P.O. Box: 400, Al-Hufof 31982, Saudi Arabia. Tel.: +966 055 8907894; fax: +966 03 5816635. E-mail address: [email protected] (S.M. El-Bahr).
(Wilson, 1984; Tibary and Anouassi, 1997), length of oestrous cycle from 11 to 30 days (Musa and Abusineina, 1978; Al-Eknah et al., 1993). As the ovulation is a response to the stimulus of mating (El wishy, 1987), the oestrous cycle has no luteal phase (Musa and Abusineina, 1978) and therefore is described as a follicular wave instead of oestrous cycle (Skidmore et al., 1995). Follicles mature in six days, maintained their size for 13 days and regressed in eight days) Musa and Abusineina, 1978). In 52%, of the animals the dominant follicle reach a mean diameter of 2.0 ± 0.1 cm before it begin to regress. In the remainder of
http://dx.doi.org/10.1016/j.anireprosci.2015.06.002 0378-4320/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: El-Bahr, S.M., et al., Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.002
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the animals (48%), the dominant follicle continue to grow to 4.2 ± 0.2 cm before regression commenced (Skidmore et al., 1996). The oocyte grows and matures in a biochemical environment (follicular fluid) that changes from small to large follicles (Leroy et al., 2004). The follicular fluid is an exudate of serum and 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 varies significantly during the growth phase of the follicle (Edwards, 1974; Zamboni, 1974; Bagavandoss et al., 1983; Wise, 1987; Gosden et al., 1988). In vitro fertilization (IVF) has the potential to overcome infertility problem (Ali et al., 2010) and low yield of embryos collected after superovulation reported in Camelidae (Anouassi and Tibary, 2013) and therefore the maturation of oocytes is one of the prerequisites of successful IVF (Wang et al., 1997). Changes in biochemical metabolites in the follicular fluid may influence oocyte maturation and quality (Richards, 1994; Hyttel et al., 1997; Gode et al., 2011; Leroy et al., 2004). The present study aimed to compare biochemical and hormonal constituents in follicular fluids and serum from female dromedary camels with different ovarian follicle sizes. 2. Materials and methods 2.1. Experimental materials One hundred ovarian pairs were recovered from clinically healthy adult (6–15 years of age) non-pregnant female camels (C. dromedarius) at a local abattoir in Kingdom of Saudi Arabia during the breeding season (November–April, 2014). Blood samples (10 ml/each animal) were collected in test tubes from all animals during exsanguinations. Preslaughter information about the reproductive status of these animals was not available. After slaughter, macroscopic examination showed clinically normal reproductive organs. Ovaries and blood samples were kept in an ice box and transported immediately (within 1 hour postslaughter) to the laboratory. Upon arrival at the laboratory, ovaries were washed twice in cooled 0.9% NaCl and blotted dry. Paired ovaries with corpus luteum were excluded from the investigation. Three different follicle classes, based on follicle diameter (measured by Vernier caliper) were considered for puncture: 1.1–1.5 cm (n = 10), 1.6–2.1 cm (n = 10) and 2.2–2.5 cm. Follicles filled with sanguineous fluid were excluded. Follicular fluids were aspirated from 10 follicles for each size by means of a sterilized 22 gauge hypodermic needles and syringes. The follicular fluid was centrifuged at 1250 × g at 4 ◦ C for 10 min. The supernatant was harvested and stored at −20 ◦ C until the time of analysis. Sera from selected animals were separated and stored at −20 ◦ C until the time of analysis. 2.2. Estimation of biochemical constituents in serum and follicular fluid Commercial diagnostic kits (United Diagnostic Industry, UDI, Dammam, Saudi Arabia) were used for determination of glucose (mg/dL; Catalog No. EP37L-660), cholesterol
(mg/dL; Catalog No. EP24-660), ACP (IU/L; Catalog No. EP02-295) and ALP (IU/L; Catalog No. EP04L-660). The analysis was conducted by using a full-automated chemistry analyzer (ELIPSE, Rome, Italy). Ascorbic acid (mM/mL) was determined using EIA kits (Catalog No. K661-100) produced by Biovision research products, Linda Vista Avenue, Mountain View, CA, USA. The procedures for analysis and calculation were adopted as recommended by the manufacturer. 2.3. Estimation of hormonal constituents of serum and follicular fluid The concentrations of E2 (pg/mL) and P4 (ng/mL) in serum and follicular fluids were analyzed using enzyme immune assay (EIA) kits (Cayman Chemical Company, Ann Arbor, MI, USA). The intra and inter assay CV’s were (5.3%, 4.7%) and (4.9%, 2.5%) for both E2 and P4 , respectively. The concentrations of T3 and T4 (g/mL) were analyzed using EIA kits obtained from BioCheck, Foster city, CA, USA. The intra and inter assay coefficient of variances (CV’s) were (4.1%, 2.3%) and (5.1%, 3.1%) for both T3 and T4 , respectively. Cortisol analysis (ng/mL) was determined by EIA kits (Oxford Biomedical Research Inc., Oxford, MI, USA). The intra and inter assay CV’s were 7.2% and 5.1%, respectively. Bovine IGF I (ng/mL) was assayed using enzyme linked immunosorbent assay (ELISA) kits (Cataloge No. MBS701164; Biosource Inc., San Diego, CA, USA) (Ghoneim et al., 2013). The intra and inter assay CV’s were 3.2% and 4%, respectively. All assays were performed according to the manufacturer’s directions, and the optical densities were measured using an ELISA reader (Absorbance Microplate Reader ELx800TM, BioTek® , Highland Park, VT, USA and Microplate Strip Washer (ELx800 TM, BioTek® , Highland Park, VT, USA). 2.4. Statistical analysis The data analysis of biochemical constituents and hormones in follicular fluid of different sized follicles and the serum was carried out using a General Linear Model (GLM) procedure and means were compared by Least Significant Difference (LSD) using SPSS 16.0 statistical software (SPSS, 2007). 3. Results Data summarized in Table 1 shows no significant differences (P > 0.05) among the values of ascorbic acid, glucose, cholesterol, ACP and ALP in follicular fluids of different sized follicles. Similarly, no significant differences (P > 0.05) were recorded in the concentrations of the same parameters in the serum of female camels with different sized follicles (Table 2). The mean concentrations of follicular hormones in different sized follicles are presented in Table 3. The concentrations of E2 in the follicular fluid of follicles sized 2.2–2.5 cm were significantly lower (P < 0.01) than its corresponding values in other follicle sizes. No significant differences were recorded (P > 0.05) in P4 , T3 , T4 , cortisol and IGF-1 concentrations of follicular fluids of all examined follicle sizes. Data presented in Table 4 shows
Please cite this article in press as: El-Bahr, S.M., et al., Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.002
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Table 1 Concentrations (mean ± SE) of different biochemical constituents in follicular fluids of different sized follicles collected from female dromedary camels. Constituents
Fluid from follicle 1.1–1.5 cm
Ascorbic acid (mM) Glucose (mg/dL) Cholesterol (mg/dL) Acid phosphatase (IU/L) Alkaline phosphatase (IU/L)
1.80 150.3 16.3 1.7 18.5
± ± ± ± ±
0.3 30.5 4.2 0.2 3.5
Fluid from follicle 1.6–2.1 cm 2.20 153.0 18.5 1.7 20.2
± ± ± ± ±
0.4 20.0 2.1 0.2 2.5
Fluid from follicle 2.2–2.5 cm 1.70 196.0 18.4 2.1 22.1
± ± ± ± ±
0.2 20.0 4.9 0.5 2.2
Table 2 Concentrations (mean ± SE) of different biochemical constituents in serum of female camels with different sized follicles. Constituents
Serum of camels with follicle 1.1–1.5 cm
Ascorbic acid (mM) Glucose (mg/dL) Cholesterol (mg/dL) Acid phosphatase (IU/L) Alkaline phosphatase (IU/L)
1.6 250.1 60.1 4.4 53.4
± ± ± ± ±
Serum of camels with follicle 1.6–2.1 cm
0.02 32.3 3.9 0.5 5.5
1.6 275.0 58.2 4.6 55.5
± ± ± ± ±
0.02 30.2 5.1 0.3 2.4
Serum of camels with follicle 2.2–2.5 cm 1.6 290.3 60.4 4.7 60.2
± ± ± ± ±
0.02 40.7 4.1 0.3 5.1
Table 3 Concentrations (mean ± SE) of selected hormones in follicular fluid of different sized follicles collected from female dromedary camels. Hormones
Fluid from follicle 1.1–1.5 cm
Estradiol 17- (pg/mL) Progesterone (ng/mL) Tri-iodothyronine (ng/mL) Thyroxin (g/mL) Cortisol (ng/mL) Insulin-like growth factor-1 (ng/mL)
157.3a 158.3 0.8 12.5 1.7 60.0
± ± ± ± ± ±
Fluid from follicle 1.6–2.1 cm 162.0a 160.2 1.0 14.0 2.0 60.0
4.4 3.1 0.2 1.5 0.3 0.5
± ± ± ± ± ±
Fluid from follicle 2.2–2.5 cm 75.6b 163.0 1.1 14.5 2.1 60.0
5.0 4.0 0.1 2.0 0.3 0.0
± ± ± ± ± ±
5.0 1.0 0.1 2.5 0.3 0.0
Means with different superscripts in the same row are significantly different at P < 0.01. Table 4 Concentrations (mean ± SE) of selected hormones in serum of female camels with different sized follicles. Hormones
Serum of camels with follicle 1.1–1.5 cm
Estradiol 17- (pg/mL) Progesterone (ng/mL) Tri-iodothyronine (ng/mL) Thyroxin (g/mL) Cortisol (ng/mL) Insulin-like growth factor-1 (ng/mL)
228.6 0.6 1.7 14.5 12.0 11.3
± ± ± ± ± ±
32.1 0.2 0.1 0.7 4.0 0.5
Serum of camels with follicle 1.6–2.1 cm 211.1 0.6 1.8 15.0 13.8 12.5
no significant variations (P > 0.05) in the concentrations of E2, P4 , T3 , T4 , cortisol and IGF-1 in the serum of all female camels with different sized follicles. However, surprisingly as presented in Table 5, mean concentrations of P4 and IGF1 in follicular fluids were higher approximately 266 and 5 folds, respectively than that in sera of female camels with
± ± ± ± ± ±
31.9 0.1 0.2 0.6 3.9 0.5
Serum of camels with follicle 2.2–2.5 cm 210.1 0.6 1.8 15.1 14.7 12.1
± ± ± ± ± ±
30.0 0.1 0.2 0.5 3.0 0.5
different sized follicle. Moreover, mean concentrations of glucose, cholesterol, ALP activity and cortisol in the sera of female camels with different sized follicle were higher approximately 1.5-, 3.5-, 2- and 7-folds, respectively than their corresponding values in follicular fluids of examined different sized follicle.
Table 5 Comparison of concentrations (mean ± SE) of selected biochemical and hormonal parameters in the follicular fluid and serum of female camel with different sized follicles. Hormones
Follicular fluid
Serum
Ascorbic acid (mM) Glucose (mg/dL) Cholesterol (mg/dL) Acid phosphatase (IU/L) Alkaline phosphatase (IU/L) Progesterone (ng/mL) Tri-iodothyronine (ng/mL) Thyroxin (g/mL) Cortisol (ng/mL) Insulin-like growth factor-1 (ng/mL)
1.9 170.1 17.0 2.2 20.0 160.0 1.0 13.0 2.0 60.0
± ± ± ± ± ± ± ± ± ±
1.6 260.1 60.0 4.5 55.0 0.6 1.6 15.0 14.0 12.0
0.2 30.0 1.0 0.5 2.0 2.0 0.1 1.0 0.3 0.0
Fold increase or decrease ± ± ± ± ± ± ± ± ± ±
0.02 30.0 2.0 0.1 5.0 0.2 0.2 1.0 1.0 1.0
– 1.5 3.5 – 2 266 – – 7 5
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4. Discussion Biochemical constituents and hormonal profiles of follicular fluids of small and large ovarian follicles have been investigated in pregnant and non-pregnant dromedary camels (Zia-Ur-Rahman et al., 2008; El-Shahat et al., 2013) during the peak and low breeding season (Ali et al., 2008, 2011). However, the current study investigated new biochemical (Ascorbic acid) and hormonal (IGF-1) alterations that have been occurred in the follicular fluid and serum of female dromedary camels (C. dromedarius) with different sized ovarian follicles. Follicular fluid contains different biochemical metabolites that derived from serum or produced locally in the follicles and shared in the metabolic activities of follicular cells (Edwards, 1974; Gérard et al., 2002). The biochemical metabolites of follicular fluids are essential for maturation and even fertilization of the oocyte. Therefore, changes of these metabolites may affect the growth and quality of the oocyte (Gode et al., 2011). The metabolic activities and properties of the blood–follicle barrier changed significantly during the follicle maturation (Bagavandoss et al., 1983; Gosden et al., 1988), ovarian diseases (Short, 1962; Boryczko et al., 1995; Khan et al., 2011) and season in seasonal breeding animals (Ali et al., 2008). The role of ascorbic acid in steroidogenesis, remodeling of follicular membrane and antioxidant system of the ovarian follicle has been reported (Goralczyk et al., 1992; Luck et al., 1995). The current study demonstrated that, the concentrations of ascorbic acid were not changed significantly neither in the follicular fluids of all examined follicle nor in the serum of female camels with different sized follicles. Parallel to the current findings, ascorbic acid concentrations were nearly the same in follicular fluids of provulatotory and oversized follicles in female dromedary camels (Ghoneim et al., 2013). Although, low ascorbic acid level was detected in the follicular fluid of cystic buffaloes (Khan et al., 2011), growth rate or morphology of the follicles were not influenced (Murray et al., 2001). Follicular glucose, the major energy source for the ovary, plays an important role in the ovarian metabolism, originates from the glycolysis at granulose cells (Leese and Lenton, 1990; Collins et al., 1997) and regulates steroidogenesis (Williams et al., 2001; Munõz-Gutiérrez et al., 2004). The current findings recorded non-significant differences in the concentrations of glucose among the follicular fluids of different follicle sizes. Similar findings (Ghoneim et al., 2013) have been reported in the follicular fluids of provulatory and oversized follicles of dromedary female camels. In the contrary, it has been reported that, glucose content in the follicular fluid of small follicles was significantly higher than its corresponding value in large follicles of female camels (Ali et al., 2008; El-Shahat et al., 2013). In dairy cows, follicular glucose concentration was directly proportional to the follicular size (Leroy et al., 2004). The current study demonstrated that, in follicular fluid, mean concentration of glucose was significantly lower than its corresponding values in the serum of camels with different sized follicles. The same results were documented previously (Zia-UrRahman et al., 2008) in female dromedary camels. Follicular cholesterol, the precursor of all steroid hormones, originates from granulosa cells and/or escape from blood
(Endresen et al., 1990). The present study reported that, the concentration of follicular cholesterol of all examined ovarian follicle were not changed significantly. Similar findings have been reported in follicular fluids of provulatory and oversized follicles of female dromedary camels (Ghoneim et al., 2013). However, the concentration of cholesterol was significantly lower in large follicles when compared with its values in the small follicles of female dromedary camels (Zia-Ur-Rahman et al., 2008). The current findings demonstrated that, the concentration of cholesterol in the serum was 3.5 folds higher than its corresponding value in the follicular fluids of all follicle sizes which agree with previous report (Zia-Ur-Rahman et al., 2008) in female dromedary camels. This may suggests that, serum cholesterol is not the major metabolic compound available for steroidogenesis in ovarian follicle. In addition, granulosa cells which have a large store of cholesteryl esters may offer free cholesterol for the preovulatory progesterone or overall steroidogenesis (Endresen et al., 1990). Phosphatase enzymes play a vital role in follicular growth and atresia and are constituents of follicular fluid of female camels (Zia-Ur-Rahman et al., 2008), humans (Caucig et al., 1972), pigs (McGaughey, 1975) and cows (Henderson and Cupps, 1990). ALP plays a role in the active transport of nutrients and secretary material across the membrane (Sangha and Guraya, 1988; Sharma, 2000). The activity of follicular ALP but not for ACP is a reliable indicator of follicular functional status (Wise, 1987). The current study revealed that, the activities of ACP and ALP remained comparable in the follicular fluid of all follicle sizes. Similar findings have been reported in follicular fluids of provulatory and oversized follicles of female dromedary camels (Ghoneim et al., 2013) and buffaloes (Khan et al., 2011). There were no significant difference in ALP and ACP activities in small and large follicles of female dromedary camels (Zia-Ur-Rahman et al., 2008). In the current study, the average concentration of E2 recorded in the sera of animals with all examined follicles was higher than the characteristic level of the estrus in dromedary camels (Elias et al., 1984). The present study reported a significant low concentration of E2 in the follicular fluid of big sized follicles (2.2–2.5 cm) compared to corresponding concentrations in other follicles sizes. Similar findings were recorded in cattle (Short, 1962; Grado-Ahuir et al., 2011) and female dromedary camels (Ghoneim et al., 2013). However, there was no significant variation in the concentration of E2 among different follicle sizes in buffaloes (Khan et al., 2011). The present study reported that, the concentration of E2 was not changed significantly in serum of camels with different sized follicles. Similar results were documented in female camels (Ghoneim et al., 2013) and cows (Nessan and King, 1981; Glencross and Munro, 1974). Granulosa and theca cells of bovine follicles produce large amounts of P4 which serves as a precursor for androgen and subsequently estrogen production (McNatty et al., 1984). The present findings demonstrated that, mean concentration of P4 in follicular fluid of different follicle sizes was not changed significantly. Similar findings have been reported in follicular fluids of provulatory and oversized follicles of female dromedary camels (Ghoneim et al., 2013). The primary source of peripheral P4 in the female dromedary camels is the corpus luteum, therefore in the absence
Please cite this article in press as: El-Bahr, S.M., et al., Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.002
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of mating and ovulation, plasma P4 level remain very low (<1 ng/mL) throughout the follicular wave (Homeida et al., 1988; Skidmore et al., 1994). Since all female camels included in the present study were non-pregnant, serum progesterone levels were low (<1 ng/mL). Parallel to the current study, it was reported that, the concentration of P4 was approximately 1000-fold in follicular fluid than serum value in Golden Hamster (Libersky and Boatman, 1995) whereas, in female dromedary camels its concentration was 300 folds in follicular fluid of oversized follicles than serum values (Ghoneim et al., 2013). The current findings revealed that, levels of T3 , T4 and cortisol in follicular fluids of all follicles were comparable. This finding agrees with previous studies in female dromedary camels (Ghoneim et al., 2013) and buffaloes (Khan et al., 2011). IGF-1 has been produced in vitro from bovine granulosa cells (Spicer et al., 1993). IGF-I regulates ovarian follicular maturation and differentiation (Adashi et al., 1985; Hammond et al., 1988). Insulin and IGF-1 stimulate Biosynthesis of E2 in granulosa cells and androgen in theca cells of bovines (Spicer et al., 1993; Butler et al., 2004). The current results recorded that, the concentration of IGF-1 in serum and follicular fluids was not influenced by follicles sizes in female dromedary camels. Previous study (Ghoneim et al., 2013) recorded that, oversized follicles in female dromedary camels were associated with decreased concentrations of IGF-I in follicular fluid, but not in serum. The current study revealed that there were no significant differences in biochemical and hormonal constituents in sera of female dromedary camels with different sized follicle. With the exception of E2, there were no significant differences in biochemical and hormonal constituents between follicular fluids from different sized follicles. Conflict of interest The authors have declared that there are no conflict of interest. Acknowledgments This research paper was supported by grants from the Deanship of Scientific Research, King Faisal University, Kingdom of Saudi Arabia. References Adashi, E.Y., Resnidc, C.E., Dercole, A.J., Svoboda, M.E., Van Wyk, J.J., 1985. Insulin like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr. Rev. 6, 400–420. Al-Eknah, M.M., Dafalla, E.A., Homeida, A.M., Galil, A.K.A., Taher, A.Y., 1993. Spontaneous uterine activity during the oestrous cycle of the camel (Camelus dromedarius). Anim. Reprod. Sci. 32, 91–97. Ali, A., Al-sobayil, F.A., Tharwat, M., Al-Hawas, A., Ahmed, A.F., 2010. Causes of infertility in female camels (Camelus dromedarius) in middle of Saudi Arabia. J. Agr. Vet. Sci. Qassim Univ. 2, 59–66. Ali, S., Ahmad, N., Akhtar, N., Zia-ur-Rahman, Ahmad, M., 2011. Hormonal profiles in the serum and follicular fluid of female camel (Camelus dromedarius) during the peak and the low breeding season. Pak. Vet. J. 31, 331–335. Ali, S., Ahmad, N., Akhtar, N., Zia-ur-Rahman, Noakes, D.E., 2008. Metabolite contents of blood serum and fluid from small and large sized follicles in dromedary camels during the peak and the low breeding seasons. Anim. Reprod. Sci. 108, 446–456.
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Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles S.M. El-Bahr a,b,∗ , I.M. Ghoneim c , M.M. Waheed c a Department of Physiology, Biochemistry and Pharmacology (Biochemistry), College of Veterinary Medicine and Animal Resources, King Faisal University, Saudi Arabia b Department of Biochemistry, Faculty of Veterinary Medicine, Alexandria University, Egypt c Department of Clinical Studies, College of Veterinary Medicine and Animal Resources, King Faisal University, Saudi Arabia
a r t i c l e
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Article history: Received 25 February 2015 Received in revised form 21 May 2015 Accepted 3 June 2015 Available online xxx Keywords: Camels Different sized follicles Follicular fluid Serum Hormones Biochemistry
a b s t r a c t The current study aimed to compare some biochemical and hormonal constituents in follicular fluids and serum of female dromedary camels with different sized ovarian follicles. Therefore, follicular fluids from follicles sized 1.1–1.5 cm (n = 10), 1.6–2.1 cm (n = 10) and 2.2–2.5 cm (n = 10) and sera were harvested from 20 female camels. The concentrations of ascorbic acid, glucose, cholesterol and activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) were not changed significantly neither in follicular fluids of all follicle sizes nor in sera of female camels with different sized follicles. The concentrations of estradiol-17 (E2) in the follicular fluid of follicles sized 2.2–2.5 cm were significantly lower (P < 0.01) than its corresponding value in follicular fluid of other follicle sizes. The concentrations of progesterone (P4 ), tri-iodothyronine (T3 ), thyroxin (T4 ), cortisol and insulin-like growth factor-1 (IGF-1) remained comparable in follicular fluids of all examined different sized follicles. The concentrations of E2, P4 , T3 , T4 , cortisol and IGF-1 were similar in the serum of camels with different sized follicles. Interestingly, mean concentrations of P4 and IGF-1 in follicular fluids were higher than their corresponding values in sera of camels with different sized follicles and the mean concentrations of glucose, cholesterol, ALP and cortisol in sera were higher than their corresponding values in follicular fluids of the examined camels. With the exception of E2, there were no significant differences in biochemical and hormonal constituents between follicular fluids from different sized follicles. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The dromedary camel (Camelus dromedarius) is a seasonal breeder with a relatively short breeding season
∗ Corresponding author at: Department of Physiology, Biochemistry and Pharmacology (Biochemistry), College of Veterinary Medicine and Animal Resources, King Faisal University, P.O. Box: 400, Al-Hufof 31982, Saudi Arabia. Tel.: +966 055 8907894; fax: +966 03 5816635. E-mail address: [email protected] (S.M. El-Bahr).
(Wilson, 1984; Tibary and Anouassi, 1997), length of oestrous cycle from 11 to 30 days (Musa and Abusineina, 1978; Al-Eknah et al., 1993). As the ovulation is a response to the stimulus of mating (El wishy, 1987), the oestrous cycle has no luteal phase (Musa and Abusineina, 1978) and therefore is described as a follicular wave instead of oestrous cycle (Skidmore et al., 1995). Follicles mature in six days, maintained their size for 13 days and regressed in eight days) Musa and Abusineina, 1978). In 52%, of the animals the dominant follicle reach a mean diameter of 2.0 ± 0.1 cm before it begin to regress. In the remainder of
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the animals (48%), the dominant follicle continue to grow to 4.2 ± 0.2 cm before regression commenced (Skidmore et al., 1996). The oocyte grows and matures in a biochemical environment (follicular fluid) that changes from small to large follicles (Leroy et al., 2004). The follicular fluid is an exudate of serum and 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 varies significantly during the growth phase of the follicle (Edwards, 1974; Zamboni, 1974; Bagavandoss et al., 1983; Wise, 1987; Gosden et al., 1988). In vitro fertilization (IVF) has the potential to overcome infertility problem (Ali et al., 2010) and low yield of embryos collected after superovulation reported in Camelidae (Anouassi and Tibary, 2013) and therefore the maturation of oocytes is one of the prerequisites of successful IVF (Wang et al., 1997). Changes in biochemical metabolites in the follicular fluid may influence oocyte maturation and quality (Richards, 1994; Hyttel et al., 1997; Gode et al., 2011; Leroy et al., 2004). The present study aimed to compare biochemical and hormonal constituents in follicular fluids and serum from female dromedary camels with different ovarian follicle sizes. 2. Materials and methods 2.1. Experimental materials One hundred ovarian pairs were recovered from clinically healthy adult (6–15 years of age) non-pregnant female camels (C. dromedarius) at a local abattoir in Kingdom of Saudi Arabia during the breeding season (November–April, 2014). Blood samples (10 ml/each animal) were collected in test tubes from all animals during exsanguinations. Preslaughter information about the reproductive status of these animals was not available. After slaughter, macroscopic examination showed clinically normal reproductive organs. Ovaries and blood samples were kept in an ice box and transported immediately (within 1 hour postslaughter) to the laboratory. Upon arrival at the laboratory, ovaries were washed twice in cooled 0.9% NaCl and blotted dry. Paired ovaries with corpus luteum were excluded from the investigation. Three different follicle classes, based on follicle diameter (measured by Vernier caliper) were considered for puncture: 1.1–1.5 cm (n = 10), 1.6–2.1 cm (n = 10) and 2.2–2.5 cm. Follicles filled with sanguineous fluid were excluded. Follicular fluids were aspirated from 10 follicles for each size by means of a sterilized 22 gauge hypodermic needles and syringes. The follicular fluid was centrifuged at 1250 × g at 4 ◦ C for 10 min. The supernatant was harvested and stored at −20 ◦ C until the time of analysis. Sera from selected animals were separated and stored at −20 ◦ C until the time of analysis. 2.2. Estimation of biochemical constituents in serum and follicular fluid Commercial diagnostic kits (United Diagnostic Industry, UDI, Dammam, Saudi Arabia) were used for determination of glucose (mg/dL; Catalog No. EP37L-660), cholesterol
(mg/dL; Catalog No. EP24-660), ACP (IU/L; Catalog No. EP02-295) and ALP (IU/L; Catalog No. EP04L-660). The analysis was conducted by using a full-automated chemistry analyzer (ELIPSE, Rome, Italy). Ascorbic acid (mM/mL) was determined using EIA kits (Catalog No. K661-100) produced by Biovision research products, Linda Vista Avenue, Mountain View, CA, USA. The procedures for analysis and calculation were adopted as recommended by the manufacturer. 2.3. Estimation of hormonal constituents of serum and follicular fluid The concentrations of E2 (pg/mL) and P4 (ng/mL) in serum and follicular fluids were analyzed using enzyme immune assay (EIA) kits (Cayman Chemical Company, Ann Arbor, MI, USA). The intra and inter assay CV’s were (5.3%, 4.7%) and (4.9%, 2.5%) for both E2 and P4 , respectively. The concentrations of T3 and T4 (g/mL) were analyzed using EIA kits obtained from BioCheck, Foster city, CA, USA. The intra and inter assay coefficient of variances (CV’s) were (4.1%, 2.3%) and (5.1%, 3.1%) for both T3 and T4 , respectively. Cortisol analysis (ng/mL) was determined by EIA kits (Oxford Biomedical Research Inc., Oxford, MI, USA). The intra and inter assay CV’s were 7.2% and 5.1%, respectively. Bovine IGF I (ng/mL) was assayed using enzyme linked immunosorbent assay (ELISA) kits (Cataloge No. MBS701164; Biosource Inc., San Diego, CA, USA) (Ghoneim et al., 2013). The intra and inter assay CV’s were 3.2% and 4%, respectively. All assays were performed according to the manufacturer’s directions, and the optical densities were measured using an ELISA reader (Absorbance Microplate Reader ELx800TM, BioTek® , Highland Park, VT, USA and Microplate Strip Washer (ELx800 TM, BioTek® , Highland Park, VT, USA). 2.4. Statistical analysis The data analysis of biochemical constituents and hormones in follicular fluid of different sized follicles and the serum was carried out using a General Linear Model (GLM) procedure and means were compared by Least Significant Difference (LSD) using SPSS 16.0 statistical software (SPSS, 2007). 3. Results Data summarized in Table 1 shows no significant differences (P > 0.05) among the values of ascorbic acid, glucose, cholesterol, ACP and ALP in follicular fluids of different sized follicles. Similarly, no significant differences (P > 0.05) were recorded in the concentrations of the same parameters in the serum of female camels with different sized follicles (Table 2). The mean concentrations of follicular hormones in different sized follicles are presented in Table 3. The concentrations of E2 in the follicular fluid of follicles sized 2.2–2.5 cm were significantly lower (P < 0.01) than its corresponding values in other follicle sizes. No significant differences were recorded (P > 0.05) in P4 , T3 , T4 , cortisol and IGF-1 concentrations of follicular fluids of all examined follicle sizes. Data presented in Table 4 shows
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Table 1 Concentrations (mean ± SE) of different biochemical constituents in follicular fluids of different sized follicles collected from female dromedary camels. Constituents
Fluid from follicle 1.1–1.5 cm
Ascorbic acid (mM) Glucose (mg/dL) Cholesterol (mg/dL) Acid phosphatase (IU/L) Alkaline phosphatase (IU/L)
1.80 150.3 16.3 1.7 18.5
± ± ± ± ±
0.3 30.5 4.2 0.2 3.5
Fluid from follicle 1.6–2.1 cm 2.20 153.0 18.5 1.7 20.2
± ± ± ± ±
0.4 20.0 2.1 0.2 2.5
Fluid from follicle 2.2–2.5 cm 1.70 196.0 18.4 2.1 22.1
± ± ± ± ±
0.2 20.0 4.9 0.5 2.2
Table 2 Concentrations (mean ± SE) of different biochemical constituents in serum of female camels with different sized follicles. Constituents
Serum of camels with follicle 1.1–1.5 cm
Ascorbic acid (mM) Glucose (mg/dL) Cholesterol (mg/dL) Acid phosphatase (IU/L) Alkaline phosphatase (IU/L)
1.6 250.1 60.1 4.4 53.4
± ± ± ± ±
Serum of camels with follicle 1.6–2.1 cm
0.02 32.3 3.9 0.5 5.5
1.6 275.0 58.2 4.6 55.5
± ± ± ± ±
0.02 30.2 5.1 0.3 2.4
Serum of camels with follicle 2.2–2.5 cm 1.6 290.3 60.4 4.7 60.2
± ± ± ± ±
0.02 40.7 4.1 0.3 5.1
Table 3 Concentrations (mean ± SE) of selected hormones in follicular fluid of different sized follicles collected from female dromedary camels. Hormones
Fluid from follicle 1.1–1.5 cm
Estradiol 17- (pg/mL) Progesterone (ng/mL) Tri-iodothyronine (ng/mL) Thyroxin (g/mL) Cortisol (ng/mL) Insulin-like growth factor-1 (ng/mL)
157.3a 158.3 0.8 12.5 1.7 60.0
± ± ± ± ± ±
Fluid from follicle 1.6–2.1 cm 162.0a 160.2 1.0 14.0 2.0 60.0
4.4 3.1 0.2 1.5 0.3 0.5
± ± ± ± ± ±
Fluid from follicle 2.2–2.5 cm 75.6b 163.0 1.1 14.5 2.1 60.0
5.0 4.0 0.1 2.0 0.3 0.0
± ± ± ± ± ±
5.0 1.0 0.1 2.5 0.3 0.0
Means with different superscripts in the same row are significantly different at P < 0.01. Table 4 Concentrations (mean ± SE) of selected hormones in serum of female camels with different sized follicles. Hormones
Serum of camels with follicle 1.1–1.5 cm
Estradiol 17- (pg/mL) Progesterone (ng/mL) Tri-iodothyronine (ng/mL) Thyroxin (g/mL) Cortisol (ng/mL) Insulin-like growth factor-1 (ng/mL)
228.6 0.6 1.7 14.5 12.0 11.3
± ± ± ± ± ±
32.1 0.2 0.1 0.7 4.0 0.5
Serum of camels with follicle 1.6–2.1 cm 211.1 0.6 1.8 15.0 13.8 12.5
no significant variations (P > 0.05) in the concentrations of E2, P4 , T3 , T4 , cortisol and IGF-1 in the serum of all female camels with different sized follicles. However, surprisingly as presented in Table 5, mean concentrations of P4 and IGF1 in follicular fluids were higher approximately 266 and 5 folds, respectively than that in sera of female camels with
± ± ± ± ± ±
31.9 0.1 0.2 0.6 3.9 0.5
Serum of camels with follicle 2.2–2.5 cm 210.1 0.6 1.8 15.1 14.7 12.1
± ± ± ± ± ±
30.0 0.1 0.2 0.5 3.0 0.5
different sized follicle. Moreover, mean concentrations of glucose, cholesterol, ALP activity and cortisol in the sera of female camels with different sized follicle were higher approximately 1.5-, 3.5-, 2- and 7-folds, respectively than their corresponding values in follicular fluids of examined different sized follicle.
Table 5 Comparison of concentrations (mean ± SE) of selected biochemical and hormonal parameters in the follicular fluid and serum of female camel with different sized follicles. Hormones
Follicular fluid
Serum
Ascorbic acid (mM) Glucose (mg/dL) Cholesterol (mg/dL) Acid phosphatase (IU/L) Alkaline phosphatase (IU/L) Progesterone (ng/mL) Tri-iodothyronine (ng/mL) Thyroxin (g/mL) Cortisol (ng/mL) Insulin-like growth factor-1 (ng/mL)
1.9 170.1 17.0 2.2 20.0 160.0 1.0 13.0 2.0 60.0
± ± ± ± ± ± ± ± ± ±
1.6 260.1 60.0 4.5 55.0 0.6 1.6 15.0 14.0 12.0
0.2 30.0 1.0 0.5 2.0 2.0 0.1 1.0 0.3 0.0
Fold increase or decrease ± ± ± ± ± ± ± ± ± ±
0.02 30.0 2.0 0.1 5.0 0.2 0.2 1.0 1.0 1.0
– 1.5 3.5 – 2 266 – – 7 5
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4. Discussion Biochemical constituents and hormonal profiles of follicular fluids of small and large ovarian follicles have been investigated in pregnant and non-pregnant dromedary camels (Zia-Ur-Rahman et al., 2008; El-Shahat et al., 2013) during the peak and low breeding season (Ali et al., 2008, 2011). However, the current study investigated new biochemical (Ascorbic acid) and hormonal (IGF-1) alterations that have been occurred in the follicular fluid and serum of female dromedary camels (C. dromedarius) with different sized ovarian follicles. Follicular fluid contains different biochemical metabolites that derived from serum or produced locally in the follicles and shared in the metabolic activities of follicular cells (Edwards, 1974; Gérard et al., 2002). The biochemical metabolites of follicular fluids are essential for maturation and even fertilization of the oocyte. Therefore, changes of these metabolites may affect the growth and quality of the oocyte (Gode et al., 2011). The metabolic activities and properties of the blood–follicle barrier changed significantly during the follicle maturation (Bagavandoss et al., 1983; Gosden et al., 1988), ovarian diseases (Short, 1962; Boryczko et al., 1995; Khan et al., 2011) and season in seasonal breeding animals (Ali et al., 2008). The role of ascorbic acid in steroidogenesis, remodeling of follicular membrane and antioxidant system of the ovarian follicle has been reported (Goralczyk et al., 1992; Luck et al., 1995). The current study demonstrated that, the concentrations of ascorbic acid were not changed significantly neither in the follicular fluids of all examined follicle nor in the serum of female camels with different sized follicles. Parallel to the current findings, ascorbic acid concentrations were nearly the same in follicular fluids of provulatotory and oversized follicles in female dromedary camels (Ghoneim et al., 2013). Although, low ascorbic acid level was detected in the follicular fluid of cystic buffaloes (Khan et al., 2011), growth rate or morphology of the follicles were not influenced (Murray et al., 2001). Follicular glucose, the major energy source for the ovary, plays an important role in the ovarian metabolism, originates from the glycolysis at granulose cells (Leese and Lenton, 1990; Collins et al., 1997) and regulates steroidogenesis (Williams et al., 2001; Munõz-Gutiérrez et al., 2004). The current findings recorded non-significant differences in the concentrations of glucose among the follicular fluids of different follicle sizes. Similar findings (Ghoneim et al., 2013) have been reported in the follicular fluids of provulatory and oversized follicles of dromedary female camels. In the contrary, it has been reported that, glucose content in the follicular fluid of small follicles was significantly higher than its corresponding value in large follicles of female camels (Ali et al., 2008; El-Shahat et al., 2013). In dairy cows, follicular glucose concentration was directly proportional to the follicular size (Leroy et al., 2004). The current study demonstrated that, in follicular fluid, mean concentration of glucose was significantly lower than its corresponding values in the serum of camels with different sized follicles. The same results were documented previously (Zia-UrRahman et al., 2008) in female dromedary camels. Follicular cholesterol, the precursor of all steroid hormones, originates from granulosa cells and/or escape from blood
(Endresen et al., 1990). The present study reported that, the concentration of follicular cholesterol of all examined ovarian follicle were not changed significantly. Similar findings have been reported in follicular fluids of provulatory and oversized follicles of female dromedary camels (Ghoneim et al., 2013). However, the concentration of cholesterol was significantly lower in large follicles when compared with its values in the small follicles of female dromedary camels (Zia-Ur-Rahman et al., 2008). The current findings demonstrated that, the concentration of cholesterol in the serum was 3.5 folds higher than its corresponding value in the follicular fluids of all follicle sizes which agree with previous report (Zia-Ur-Rahman et al., 2008) in female dromedary camels. This may suggests that, serum cholesterol is not the major metabolic compound available for steroidogenesis in ovarian follicle. In addition, granulosa cells which have a large store of cholesteryl esters may offer free cholesterol for the preovulatory progesterone or overall steroidogenesis (Endresen et al., 1990). Phosphatase enzymes play a vital role in follicular growth and atresia and are constituents of follicular fluid of female camels (Zia-Ur-Rahman et al., 2008), humans (Caucig et al., 1972), pigs (McGaughey, 1975) and cows (Henderson and Cupps, 1990). ALP plays a role in the active transport of nutrients and secretary material across the membrane (Sangha and Guraya, 1988; Sharma, 2000). The activity of follicular ALP but not for ACP is a reliable indicator of follicular functional status (Wise, 1987). The current study revealed that, the activities of ACP and ALP remained comparable in the follicular fluid of all follicle sizes. Similar findings have been reported in follicular fluids of provulatory and oversized follicles of female dromedary camels (Ghoneim et al., 2013) and buffaloes (Khan et al., 2011). There were no significant difference in ALP and ACP activities in small and large follicles of female dromedary camels (Zia-Ur-Rahman et al., 2008). In the current study, the average concentration of E2 recorded in the sera of animals with all examined follicles was higher than the characteristic level of the estrus in dromedary camels (Elias et al., 1984). The present study reported a significant low concentration of E2 in the follicular fluid of big sized follicles (2.2–2.5 cm) compared to corresponding concentrations in other follicles sizes. Similar findings were recorded in cattle (Short, 1962; Grado-Ahuir et al., 2011) and female dromedary camels (Ghoneim et al., 2013). However, there was no significant variation in the concentration of E2 among different follicle sizes in buffaloes (Khan et al., 2011). The present study reported that, the concentration of E2 was not changed significantly in serum of camels with different sized follicles. Similar results were documented in female camels (Ghoneim et al., 2013) and cows (Nessan and King, 1981; Glencross and Munro, 1974). Granulosa and theca cells of bovine follicles produce large amounts of P4 which serves as a precursor for androgen and subsequently estrogen production (McNatty et al., 1984). The present findings demonstrated that, mean concentration of P4 in follicular fluid of different follicle sizes was not changed significantly. Similar findings have been reported in follicular fluids of provulatory and oversized follicles of female dromedary camels (Ghoneim et al., 2013). The primary source of peripheral P4 in the female dromedary camels is the corpus luteum, therefore in the absence
Please cite this article in press as: El-Bahr, S.M., et al., Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.002
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of mating and ovulation, plasma P4 level remain very low (<1 ng/mL) throughout the follicular wave (Homeida et al., 1988; Skidmore et al., 1994). Since all female camels included in the present study were non-pregnant, serum progesterone levels were low (<1 ng/mL). Parallel to the current study, it was reported that, the concentration of P4 was approximately 1000-fold in follicular fluid than serum value in Golden Hamster (Libersky and Boatman, 1995) whereas, in female dromedary camels its concentration was 300 folds in follicular fluid of oversized follicles than serum values (Ghoneim et al., 2013). The current findings revealed that, levels of T3 , T4 and cortisol in follicular fluids of all follicles were comparable. This finding agrees with previous studies in female dromedary camels (Ghoneim et al., 2013) and buffaloes (Khan et al., 2011). IGF-1 has been produced in vitro from bovine granulosa cells (Spicer et al., 1993). IGF-I regulates ovarian follicular maturation and differentiation (Adashi et al., 1985; Hammond et al., 1988). Insulin and IGF-1 stimulate Biosynthesis of E2 in granulosa cells and androgen in theca cells of bovines (Spicer et al., 1993; Butler et al., 2004). The current results recorded that, the concentration of IGF-1 in serum and follicular fluids was not influenced by follicles sizes in female dromedary camels. Previous study (Ghoneim et al., 2013) recorded that, oversized follicles in female dromedary camels were associated with decreased concentrations of IGF-I in follicular fluid, but not in serum. The current study revealed that there were no significant differences in biochemical and hormonal constituents in sera of female dromedary camels with different sized follicle. With the exception of E2, there were no significant differences in biochemical and hormonal constituents between follicular fluids from different sized follicles. Conflict of interest The authors have declared that there are no conflict of interest. Acknowledgments This research paper was supported by grants from the Deanship of Scientific Research, King Faisal University, Kingdom of Saudi Arabia. References Adashi, E.Y., Resnidc, C.E., Dercole, A.J., Svoboda, M.E., Van Wyk, J.J., 1985. Insulin like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr. Rev. 6, 400–420. Al-Eknah, M.M., Dafalla, E.A., Homeida, A.M., Galil, A.K.A., Taher, A.Y., 1993. Spontaneous uterine activity during the oestrous cycle of the camel (Camelus dromedarius). Anim. Reprod. Sci. 32, 91–97. Ali, A., Al-sobayil, F.A., Tharwat, M., Al-Hawas, A., Ahmed, A.F., 2010. Causes of infertility in female camels (Camelus dromedarius) in middle of Saudi Arabia. J. Agr. Vet. Sci. Qassim Univ. 2, 59–66. Ali, S., Ahmad, N., Akhtar, N., Zia-ur-Rahman, Ahmad, M., 2011. Hormonal profiles in the serum and follicular fluid of female camel (Camelus dromedarius) during the peak and the low breeding season. Pak. Vet. J. 31, 331–335. Ali, S., Ahmad, N., Akhtar, N., Zia-ur-Rahman, Noakes, D.E., 2008. Metabolite contents of blood serum and fluid from small and large sized follicles in dromedary camels during the peak and the low breeding seasons. Anim. Reprod. Sci. 108, 446–456.
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Please cite this article in press as: El-Bahr, S.M., et al., Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.002