Effects of sodium selenite, cysteamine, bacterially synthesized Se-NPs, and cysteamine loaded on Se-NPs on ram sperm cryopreservation.

During the cryopreservation of sperm, the production of highly reactive oxygen species (ROS) can reduce their viability and fertility. However, the addition of antioxidants can help reduce the harmful effects of ROS. One such antioxidant is selenium, which is a co-factor of the glutathione peroxidase enzyme that is effective in scavenging ROS. Cysteamine can also take part in the structure of this enzyme. The use of nanoparticles can be less toxic to cells than their salt form. To this end, researchers synthesized Se-NPs using the streptococcus bacteria and loaded cysteamine onto the synthesized Se-NPs. The biosynthesis of Se-NPs and cysteamine loaded on Se-NPs was confirmed by UV-visible spectroscopy, X-ray diffraction (EDX), Fourier transforms infrared (FTIR) spectroscopy, and Field Emission Scanning Electron Microscope (FE-SEM). For cryopreservation, ram semen samples were diluted, and different concentrations (0, 1, 5, 25, and 125 µg/mL) of cysteamine, Se-NPs, cysteamine loaded on Se-NPs, and sodium selenite were added. An extender containing no supplement was considered as control group. After cooling the semen samples, they were frozen and stored in liquid nitrogen for evaluation. The samples were thawed and analyzed for mobility, viability, membrane and DNA integrity, and sperm abnormalities, as well as malondialdehyde level (MDA) and superoxide dismutase (SOD). The data was processed using SPSS, and a significance level of p < 0.05 was considered. The results of this experiment showed that adding 1 µg/mL of cysteamine loaded on Se-NPs to the diluent significantly increased the motility, viability, and membrane integrity and SOD of spermatozoa compared to the other treatment groups and control group, and reduced the abnormality, apoptosis, and MDA level of spermatozoa in comparison with the other treatment groups and control group (p < 0.05). In conclusion, the addition of cysteamine loaded on Se-NPs was found to improve the quality of ram sperm after cryopreservation.

The ND1 and CYTB genes polymorphisms associated with in vitro early embryo development of Sanjabi sheep.

This study aimed to investigate the association between polymorphisms of ND1 and CYTB genes and in vitro early embryo development of Sanjabi sheep. Blood and ovarian samples were collected from a local slaughterhouse. The cumulus-oocyte complexes with a diameter greater than 3 mm were aspirated from follicles, and in vitro maturation (IVM) and in vitro culture (IVC) rates of them were recorded. A respective 1200 bp and 980 bp fragments of ND1 and CYTB genes were genotyped using a modified single strand conformation polymorphism (SSCP) method. The results of this study revealed that four different patterns, named as A, B, C, and D were observed for both ND1 and CYTB genes. The ND1 gene polymorphisms had significant effects on the IVM and IVC rate (p < 0.05). The pattern C of the ND1 gene significantly increased the IVM rate compared to the patterns A, B and D. For the IVC, the highest and lowest means were related to the C and B patterns, respectively. The CYTB gene polymorphisms also had significant effects on IVC (p < 0.01), but the IVM did not affected (p = 0.07). Here, the pattern D had the highest and the pattern C had the lowest means for both IVM and IVC rates.

Pre-treatment of ram semen extender with magnetic nanoparticles on freeze-thawed spermatozoa.

BACKGROUND: Extensive use of different nanoparticles caused significant concerns about their biological safety. OBJECTIVE: This study aimed to evaluate the effects of cryopreservation on ram semen after adding magnetic nanoparticles (MNPs) to separate X and Y chromosome-bearing spermatozoa. METHODS: The experimental ram sperms in this research included treated spermatozoa (50 µg/ml MNPs) and non-treated spermatozoa. DNA damage of spermatozoa was examined using an acridine orange (AO) assay. Sperm viability, membrane functionality, abnormality and malondialdehyde (MDA) level were also measured. RESULTS: Results indicated that the pre-treatment of ram semen extender with MNPs did not significantly affect the semen parameters such as viability, membrane functionality, abnormality, as well as lipid peroxidation (LPO) levels and DNA integrity in comparison with the control group (p < 0.05). CONCLUSIONS: These observations suggest that pre-treatment of ram semen extender with MNPs after semen sexing did not have adverse effects on different semen parameters after cryopreservation.

The relationship between mitochondrial ND5 gene polymorphisms and in vitro embryo production in Sanjabi sheep.

The aim of this study was to investigate mitochondrial ND5 gene polymorphisms and their relationship with in vitro maturation (IVM) and in vitro culture (IVC) of Sanjabi sheep. Blood and ovarian samples of adult ewes were obtained from a local slaughterhouse. For each ovarian sample, cumulus-oocyte complexes larger than 3 mm in diameter were aspirated from follicles, and their IVM and IVC rates were recorded. A 666-bp fragment of the ND5 gene was amplified using the polymerase chain reaction. The samples were genotyped using a modified single-stranded conformation polymorphism (SSCP) method, and an association study was conducted with IVM and IVC rates. Six different SSCP patterns, designated A, B, C, D, E and F with respective frequencies of 8, 47, 4, 4, 32 and 5%, respectively, were observed. According to the results of association analysis, there was no significant association between the ND1 gene polymorphisms and the IVM and IVC rates (P > 0.05).

Effects of different concentrations of Chir98014 as an activator of Wnt/beta-catenin signaling pathway on oocyte in-vitro maturation and subsequent embryonic development in Sanjabi ewes.

The present study was conducted to investigate the effects of the activator factor of the WNT pathway, chir98014, leading to the in vitro sheep oocyte maturation medium, on the cumulus cell development, different nuclear maturation stages and the following process of embryonic development. Experiments included (a) addition of different concentrations (0, 0.1, 0.5, 1 µm) of chir98014 to the maturation medium and evaluation of the cumulus cell expansion, (b) addition of different concentrations of chir98014 to the maturation medium and investigation of different nuclear maturation stages, (c) addition of different concentrations of chir98014 to the maturation medium and examination of the subsequent embryonic maturation process and (d) addition of different concentrations of chir98014 to the embryonic development culture medium (the first 48 hr) and investigation of the subsequent embryonic development process. The extracted data were analysed using the SPSS software, considering the significance level of p < .05 and making the mean comparisons. The results showed that the addition of the 0.1 µM concentration of chir98014 to the maturation medium had no significant effects on the oocyte maturation and embryo development post-fertilization but it enhanced the Cumulus-oocyte complexes (COCs) expansion. In the fourth experiment, the low concentration of chir98014 in the embryo culture media improved the embryo development process, whereas the high one had a detrimental effect on it, as compared to the control group. Thus, the presence of the lower concentrations of this compound in the embryonic culture medium had favourable effects on the development of embryos.

Recovery of sperms bearing X chromosomes with different concentrations of magnetic nanoparticles in ram.

Pre-conceptual sex selection is still a highly debatable process whereby X and Y chromosome bearing spermatozoa are isolated before oocyte fertilization. Recently, magnetic nanoparticles (MNP) have been used to determine X and Y chromosomes bearing spermatozoa as a result of searching for a cheap, highly efficient method using non-toxic materials. This study aimed to recover the sperm bearing X chromosomes in ram with different concentrations of MNP and then evaluate the success of this method using polymerase chain reaction (PCR). Ram sperms were divided into four groups, treated with 0 (control), 50, 100 and 200 µg/ml MNP, respectively. MNP was used to restore sperm cells bearing X chromosomes. Upon recovery, the PCR was performed to identify the X and Y sperms, Methyl ThiazoleTetrazolium (MTT), to assess MNP toxicity and sperm viability and acridine orange (AO) to evaluate sperm DNA integrity. The results of PCR revealed that the treatment of spermatozoa- bearing X chromosomes with 50 µg/ml MNP had the highest effects on the recovery of X sperm rather than the other concentrations of MNP. However, the concentrations of MNP did not have any toxic effects on spermatozoa, sperm viability and, DNA integrity, but the high concentration of MNP (200 µg/ml) significantly reduced DNA integrity. According to MTT and AO results, the concentrations of MNP used in this study had no toxic effects on spermatozoa and did not reduce the sperm viability and DNA integrity, except that 200 µg/ml MNP significantly reduced DNA integrity.

In vitro versus In vivo: Development-, Apoptosis-, and Implantation- Related Gene Expression in Mouse Blastocyst.

BACKGROUND: While mammalian embryos can adapt to their environments, their sensitivity overshadows their adaptability in suboptimal in vitro conditions. Therefore, the environment in which the gametes are fertilized or to which the embryo is exposed can greatly affect the quality of the embryo and consequently its implantation potential. OBJECTIVES: Since providing an optimal culture condition needs a deep understanding of the environmental effects, and regarding the fact that normal morphology fails to be a reliable indicator of natural embryo development, the current study aimed at comparing in vivo- and in vitro-derived blastocysts at the molecular level. MATERIALS AND METHODS: In vivo and in vitro mouse blastocysts were obtained by flushing the uterine horns and in vitro fertilization/culture, respectively. Normal blastocysts of both groups were evaluated in terms of hatching rate and expression of three lineage-differentiation-, apoptosis-, and implantation-related genes. RESULTS: The hatching rate was lower in In vitro fertilization (IVF)-produced blastocysts in comparison with that of the in vivo counterparts. More importantly, the study results indicated significant changes in the expression levels of eight out of ten selected genes, especially Mmp-9 (about -10.7-fold). The expression of Mmp-9 in trophoblast cells is required for successful implantation and trophoblast invasion. CONCLUSIONS: The current study, in addition to confirming that the altered gene expression pattern of in vitro-produced embryos resulted in normal morphology, provided a possible reason for lower implantation rate of in vitro-produced blastocysts regarding the Mmp-9 expression.

Different Origin, Different Response: Gene Expression Pattern in Collapsed Vitrified Blastocyst.

There is a large body of animal experimental data about assisted reproductive techniques that could be applied to improve clinical outcomes. The great part of this information was obtained from research on in vivo-derived embryos. But whether these results are always similar with those we expect from embryos having in vitro origin in the clinical cases is a critical question. The present study was designed to compare the effects of vitrification (VIT) and artificial collapse (AC) as two commonly used techniques on in vivo- and in vitro-derived mouse embryos. In this regard, both origins of blastocysts were produced and randomly divided into three experimental groups, including control (non-vitrified), VIT, and AC-VIT. The survival and hatching rates and the expression of development-related genes were assessed in all groups and compared with their control counterpart. According to our results, although in vivo and in vitro origins followed the same pattern in the hatching rate, the real-time PCR data showed two distinct patterns of gene expression. Compared to the control, vitrification increased the expression of pluripotency genes in in vivo group. While in vitro vitrified blastocysts showed a significant reduction in the transcripts of these genes. More interestingly, although AC resulted in a sharp decrease of Gata6 and Grb2 in post warmed in vivo blastocysts, it could not affect the vitrified IVP ones. In conclusion, it seems that vitrification and artificial collapse techniques have different effects on embryo fate depending on in vivo or in vitro origins of the embryos.

Retraction : Resveratrol treatment during in vitro maturation enhances cumulus expansion and developmental competence of Sanjabi sheep oocytes.

This article released online on January 18, 2019 as advance publication was withdrawn from consideration for publication in The Journal of Reproduction and Development at author's request.

Retraction: Resveratrol treatment during in vitro maturation enhances cumulus expansion and developmental competence of Sanjabi sheep oocytes.

This article released online on January 18, 2019 as advance publication was withdrawn from consideration for publication in The Journal of Reproduction and Development at author's request.

The effects of sericin on cryopreserved sperm cells and subsequent embryo development in mice.

BACKGROUND: Sericin, because of its ability to remove free radicals and its antioxidant properties, has been used to successfully cryopreserve various mammalian cell types. However, the effects of sericin on cryopreservation of mouse sperm has not been reported. OBJECTIVE: The current study intended to determine the protective role of different concentrations of sericin (0, 0.25, 0.5, and 0.75%) on mouse spermatozoa during cryopreservation, in addition to its effect on in vitro fertilization and subsequent embryo development. MATERIALS AND METHODS: Mouse sperm from epididymides were frozen in cryoprotective agent with 18% raffinose, 3% skim milk, and different concentrations of sericin (0, 0.25, 0.5, 0.75%). Thawed sperm were used for in vitro fertilization. The obtained embryos were cultured in Ksom medium for 6 days. The post-thawed motility, viability, fertilizing ability, and subsequent development to the 2-cell embryo and blastocyst stages were evaluated. RESULTS: Our findings show that frozen-thawed sperm cells with 5% sericin indicate significantly (p≤0.0001) percentages of survivability and motility, the best fertilizing ability, as well as 2-cell embryo and blastocyst development compared to the other treated groups. There was no significant difference in survivability (p=0.8781), fertilizing ability (p=0.2458) and development of 2-cell (p=0.5136) and blastocysts embryos (p=0.0896) between 0.75% sericin and control groups. CONCLUSION: Supplementation by 0.5% sericin in cryoprotective agent improved frozen-thawed mouse epididymal sperm cell quality and resulted in increased embryo development.

The protease inhibitor antipain has a beneficial synergistic effect with trehalose for ram semen cryopreservation.

The objective of this study was to examine the different concentrations of antipain and trehalose combination on post-thawed quality of ram semen cryopreserved in tris extender. Ejaculates were collected from four rams using the artificial vagina, pooled at 37°C and diluted with (A0  Tre0 : antipain 0 µM and trehalose 0 mM (Control); A10  Tre0 ; A50  Tre0 ; A0  Tre30 ; A0  Tre60 ; A10  Tre60 ; A10  Tre30 ; A50  Tre30 and A50  Tre60 ). Diluted semen samples were gradually cooled down from 37 to 5°C in a cold cabinet; then, they were loaded into 0.25 ml straws, frozen and stored in liquid nitrogen. Sperm motility (CASA), viability, membrane functionality and abnormality were evaluated after thawing process. Progressive motility in extender supplemented with A10  Tre0 , A0  Tre30 and A10  Tre60 significantly (p < 0.05) higher as compared to the control (A10  Tre0 ). A10  Tre60 (47.50 ± 0.73) provided the best maintenance of progressive motility in comparison with the control (40.50 ± 0.73). No significant differences were observed between all treated groups in terms of total motility, VAP, VSL, VCL, ALH, BCF, STR and LIN. The percentages of sperm with viable were significantly higher in extenders supplemented with A10  Tre0 , A50  Tre0 , A0  Tre30 and A10  Tre60 , compared to control. Addition of A10  Tre0 , A50  Tre0 and A10  Tre60 to extenders improved the percentages of sperm abnormality, compared to the controls. A10  Tre60 (67.84 ± 1.51) treatment provided the best maintenance of normal morphology compared to the other treatments. The supplementation with A10  Tre0 , A0  Tre60 and A10  Tre60 improved the percentage of sperm membrane functionality when compared to the control (p < 0.05). Comparing these results with those of control diluents, the effects of supplementation were better except for A50  Tre60 group. In conclusion, when combination of antipain (10 µM) and trehalose (30 and 60 mM) was added, they conferred a great cryosurvival capacity with their synergic effects during freeze-thawing process.

Identification of g.170G>A and g.332G>A mutations in exon 3 of leptin gene (Bcnl and Cail) and their association with semen quality and testicular dimensions in Sanjabi rams.

The purpose of this study was to investigate leptin gene polymorphisms and their relationships with the characteristics of sperm quality and testicular dimensions. Semen samples were collected from 96 Sanjabi rams during autumn and spring seasons over two years. Simultaneously, the dimensions of length, width and scrotal circumference were measured. Blood samples were taken from the jugular vein to extract DNA. PCR was performed to amplify a 463bp fragment including exon 3 of leptin gene. PCR products were digested by Bcnl and Cail restriction enzymes to identify 170G>A and 332G>A mutations in exon 3, respectively. Leptin gene polymorphism in 170G>A locus had an effect on individual motility trait, water test and scrotal circumference (P<0.05) and animals with the AA genotype had the highest individual motility compared with the GG and GA genotypes (P<0.05). The AG genotypes had the highest water test compared with the GG and AA genotypes (P<0.05) but GG genotype had higher scrotal circumference than that of GA and AA genotypes (P<0.05). The results showed that polymorphism in 332G>A locus had a significant effect on viability trait, water test and scrotal circumference as GA genotypes had the highest amounts for these traits compared with GG genotypes (P<0.05). Based on our knowledge, the current study is the first report on the association of leptin gene polymorphisms with sperm fertility and testicular dimensions in sheep, which suggests leptin gene as a potential gene to be used in breeding programs in order to improve fertility in herds.

Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts.

Artificial collapse of the blastocoel cavity before vitrification can improve the quality of warmed embryos, yet how reduction of blastocoel fluid impacts formation of the blastocyst cell lineages is not clear. The present study assessed the effect of pre-vitrification blastocoel fluid reduction on the survival, hatching rate, and the expression of genes related to apoptosis (Tp53), pluripotency (Pou5f1, Nanog), and differentiation (Cdx2, Eomes, Gata6) in mouse blastocysts. In vivo-produced blastocysts were randomly divided into three groups: The first group was vitrified and warmed; the second group underwent artificial collapse of the blastocoel cavity prior to vitrification and warming; the third group served as the control, in which neither vitrification or artificial collapse was performed. The survival rate of treatment groups was similar to the control group, whereas the hatching rate of artificial collapse/vitrified blastocysts was significantly higher than vitrified blastocysts. Quantitative reverse-transcription PCR analysis revealed a considerable reduction in the expression of Cdx2, Eomes, Gata6, Grb2, and Tp53 transcripts following artificial collapse/vitrification in comparison to the vitrification-alone group; the abundance of Pou5f1 and Nanog, however, did not change. These results suggest that artificial collapse of the blastocoel cavity before vitrification leads to relatively normal expression of apoptosis and development-related genes plus higher hatching rates. Mol. Reprod. Dev. 83: 735-742, 2016 © 2016 Wiley Periodicals, Inc.

The presence of corpus luteum may have a negative impact on in vitro developmental competency of bovine oocytes.

The aim of the current study was to investigate the effects of the presence or absence of corpus luteum (CL) on in vitro developmental competence of bovine oocytes. In experiment 1, cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and divided according to the presence (CL(+) oocytes) or absence (CL(-) oocytes) of a CL in the ovary. Control oocytes (C group) were obtained from ovaries which were not selected toward the presence or absence of CL. All oocytes were submitted to in vitro maturation, fertilization and culture. In experiment 2, the oocytes from the CL(+) and CL(-) ovaries were divided into grown (BCB(+)) and growing (BCB(-)) categories by means of the brilliant cresyl blue (BCB) test. The oocytes from all groups (CL(+)/BCB(+), CL(-)/BCB(+), CL(+)/BCB(-), CL(-)/BCB(-) and control oocytes) were subjected to in vitro embryo production. In experiment 1, the cleavage and blastocyst rates of CL(-) oocytes were higher than those of CL(+) oocytes (83.9% and 43% vs. 69.3% and 22.5%, respectively). In experiment 2, there was less BCB(+) oocytes (more competent oocytes) in the group of CL(+) oocytes than in the group of CL(-) oocytes. Furthermore, developmental competence of all CL(+) oocytes (CL(+)/BCB(+) and CL(+)/BCB(-)) was lower than that of all CL(-) oocytes (CL(-)/BCB(+) and CL(-)/BCB(-)). Thus, the presence of a corpus luteum in the ovary may have negative effects on developmental competence of ipsilateral oocytes.

In vitro culture medium (IVC) supplementation with sericin improves developmental competence of ovine zygotes.

This study was carried out to investigate the effects of supplementation of potassium simplex optimized medium (KSOM-aa) with various sericin concentrations (0, 0.1, 0.5, 1 and 2.5%) on ovine zygotes. The results indicate that the supplementation of oocyte in vitro culture medium with optimal concentration of sericin (0.1 and 0.5%) may have beneficial effects on developmental competence of in vitro-derived ovine embryos.

In vitro developmental competence of bovine oocytes: Effect of corpus luteum and follicle size.

BACKGROUND: Previous studies reported many discrepancies about the effects of corpus luteum (CL) and ovarian follicle size on the developmental competence of oocytes. OBJECTIVE: The aim of this study was to investigate the effects of CL and different size of follicle on the developmental potential of bovine oocytes. MATERIALS AND METHODS: After ovarian classification based on presence or absence of CL, sample follicles were placed in three groups according to their diameter; small (S; 3-6 mm), medium (M; 6-9 mm), and large (L; 10-20 mm). Collected oocytes in each group were subjected to the in vitro embryo production processes. RESULTS: Results showed that, the percentages of blastocyst obtained from oocytes originating from small and medium follicles of ovaries bearing a CL (CL+S-oocytes and CL+M-oocytes, respectively) were lower (p<0.001) than those of small and medium follicles of ovaries not bearing a CL (CL-S-oocytes and CL-M-oocytes, respectively) (30.8% and 33.6% vs. 36.9% and 38.7% respectively). Although, the percentages of blastocyst obtained from CL-M-oocytes and CL-L-oocytes were greater (p< 0.001) than those of CL+S-oocytes and CL+M-oocytes. There were no significant differences in the percentages of blastocyst formation between controls (C-oocytes), CL-S-oocytes and CL+L-oocytes. CONCLUSION: According to the results of this study, the negative effect of CL on the developmental competence of bovine oocyte depends on the follicle size. Therefore, oocytes originating from large grown follicles were not influenced by negative effects of CL as much as those originating from small and medium follicles did.

In vivo and in vitro study of the function of the left and right bovine ovaries.

Inequality in function of the left and right bovine ovaries and uterine horns was evaluated in two separate experiments. In the first experiment (in vivo), the relationship between the left and right ovarian activities and reproductive indices was evaluated. Therefore, the total number of 1284 randomly chosen lactating dairy cows were examined from Day 50 to 60 postpartum, and according to the presence of an active CL on the ovaries, they were divided into 502 LCL3-cows and 782 RCL3-cows (cows with an active CL on the left [L] or right [R] ovary, respectively). To induce estrus synchronization and investigate the effects of PGF2α administration on the incidence of estrus in both LCL3-cows and RCL3-cows, the cows were treated with one luteolytic dose of PGF2α and were inseminated after observed estrus (via visual observation lasting at least 30 minutes three times a day). To investigate the effects of side of ovulation at the time of PGF2α administration on reproductive parameters, pregnancy diagnosis was performed 28 days after insemination (using ultrasound) and 42 days after insemination (using transrectal palpation). The results showed that the percentage of the RCL3-cows was greater than the LCL3-cows (60.9% vs. 39.1%, respectively). Furthermore, ovulations switching from the left to right ovary in two successive ovulations were greater than those that switched from the right to left ovary. On the other hand, the sex ratio (male percentage) in the right uterine horn was greater than that of the left one. In the second experiment (in vitro), the developmental potential of bovine oocytes derived from the left (L-oocytes) and right (R-oocytes) ovaries after in vitro embryo production and heterogeneity in the developmental competence of L-oocytes and R-oocytes using the brilliant cresyl blue staining test as a selection criterion were evaluated. Results of the in vitro experiment showed that the percentage of cleavage and blastocyst rate of R-oocytes were greater (P < 0.001) than those of L-oocytes. Moreover, it appears that the side of ovaries had greater effects on the developmental competence of oocytes than other factors associated with heterogeneity in the developmental competence of oocytes, which can be detected by the brilliant cresyl blue test. In conclusion, the results of the in vivo study confirmed the observations in previous studies in which the right ovarian response (distribution of ovulation) was superior to that of the left ones. Interestingly, the in vitro experiments for the first time clearly showed that more ovulation on the right side is not the only reason for this unequal activity. In fact, in cattle, the greater developmental potential of oocytes originating from right ovaries may cause superior activity of the right side, and the effect is even higher than the differences in ovulation response between the left and right ovaries.

The influence of the corpus luteum on metabolites composition of follicular fluid from different sized follicles and their relationship to serum concentrations in dairy cows.

The presence of corpus luteum may have a local effect on metabolite composition of follicular fluid (FF) and could indirectly influence follicular development and oocyte quality. The purpose of this study was to examine the influence of the corpus luteum on metabolite composition of follicular fluid (FF), harvested from different-sized follicles and the relationship between metabolite composition of FF to blood serum in dairy cows. Ovaries and blood samples were collected from 30 female adult Holstein Friesian cows, 4-7 years old, with clinically normal reproductive tracts. The animals were in the diestrus stage and selected post mortem. The ovaries collected were classified based on the presence and absence of corpus luteum (CL(+/-)). Visible follicles on the surface of the ovaries were classified into (i) small (3-5mm), (ii) medium (6-9 mm) and (iii) large (10-20mm) based on their diameter. Follicular fluid was aspirated from follicles with different sizes in CL(+) and CL(-) ovaries. Blood and FF samples were analyzed for various biochemical constituents including glucose, cholesterol, triglyceride, total protein, albumin and globulin. The results showed that serum concentration of glucose, cholesterol and triglyceride was significantly different (p≤0.05) in FF from follicles of different size categories. Differences between various follicle size categories in CL(-) ovaries were only significant for concentrations of glucose, cholesterol and triglyceride. FF concentration of glucose and cholesterol in the same follicle size categories in CL(+) ovaries was significantly lower than that of CL(-) ovaries. These results indicate that levels of the biochemical metabolites in serum and FF differ significantly. In addition, FF concentrations of biochemical metabolites were related to follicular size and to the presence or absence of corpus luteum.