Buffalo Follicular Fluid Research Articles

Development of buffalo embryo derived from ICSI: effects of various somatic-cell co-culture

Context Somatic-cell co-culture of intracytoplasmic sperm injection (ICSI) buffalo embryos has not been reported earlier. Aim This study aimed to determine the effects of buffalo oviductal epithelial-cell, granulosa-cell, and cumulus-cell co-culture on in vitro culture of early embryo development as ICSI and post-activation. Methods Selected oocyte–cumulus complexes were cultured for 19–20 h in 50-μL drop of tissue culture medium (TCM199 + 10% buffalo follicular fluid, hCG 50 IU/mL, 0.02% arbitrary units (AU)/mL follicle-stimulating hormone and 1 μg/mL estradiol-17βE2). Oocytes reaching Metaphase II were subjected to ICSI with immobilised spermatozoa. All ICSI oocytes were activated with calcium ionophore for 5 min, followed by cycloheximide for 5 h. The embryos at 6–8-cell stages were co-cultured. Key results The morula, blastocyst, and hatched blastocyst rates when co-cultured with oviductal epithelial cells were 68.18%, 48.18%, and 30.00% respectively. The morula, blastocyst, and hatched blastocyst rates when co-cultured with cumulus cells were 51.49%, 34.33%, and 16.42% respectively. The morula, blastocyst, and hatched blastocyst rates when co-cultured with granulosa cells were 52.14%, 32.48%, and 13.68% respectively. Conclusions In vitro maturation buffalo oocytes can be fertilised in vitro with ICSI and co-cultured with different types of cells. Oviductal epithelial cell co-culture was shown to be superior in supporting in vitro embryo development in this study. Implications The oviductal epithelial cells are easy to prepare and may be used for co-culture to increase the efficiency of in vitro production of buffalo embryos.

Assessment Viability of Frozen-Thawed Oocytes Using Non- Invasive Technique based on Buffalo Follicular Fluid

Assessment Viability of Frozen-Thawed Oocytes Using Non- Invasive Technique based on Buffalo Follicular Fluid

Association of taurine with ovarian follicular steroids and postpartum anestrus condition in Murrah buffaloes

Taurine is an abundant intracellular beta-amino acid majorly synthesized in the liver and transported through plasma. In mammals, taurine was reported to be involved in various physiological functions, including the enhancement of testosterone levels, the major estradiol precursor. Therefore, we hypothesize that taurine levels are associated with ovarian follicular steroids as well as with a reproductive problem called postpartum anestrus (PPA) in dairy buffaloes. To understand the taurine levels and its possible role in buffalo ovarian follicles, a correlation was established among taurine, estradiol, and testosterone levels in the ovarian follicular fluid. For this purpose, buffalo ovaries were obtained from the slaughterhouse, and follicular fluid samples were collected from small (<4 mm), medium (4–8 mm) and large (>8 mm) follicles. Taurine and steroid levels in the follicular fluid were analyzed by TLC and ELISA, respectively. Taurine and testosterone levels were significantly (P < 0.05) higher in the follicular fluid of small and medium follicles than large follicles, whereas the estradiol levels were significantly (P < 0.001) higher in the large follicles. Thus, taurine showed a positive correlation (r = 0.75) with testosterone and a negative correlation (r = −0.77) with estradiol in buffalo follicular fluid, indicating its possible role in testosterone function during follicular development. Interestingly, significantly (P < 0.001) lower plasma taurine levels in PPA (n = 50) than normal cyclic (n = 50) buffaloes represented its association with PPA. Therefore, our present study recommends the need for future nutrition studies on taurine supplementation to PPA buffaloes.

Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

Biochemical and Fatty Acids Composition of Water Buffalo (Bubalus Bubalis) Follicular Fluid

Aim of this study was to characterize the biochemical and fatty acids composition of follicular fluid collected from follicles of different sizes and in different phases of ovarian cycle in water buffalo farmed in Italy. Ovaries were collected at slaughterhouse during the breeding season; follicular fluid was aspirated dividing samples in small and large follicles (&lt; 6 mm and &gt; 6 mm respectively) and in luteal and follicular phase. Biochemical analysis and gas-chromatography were performed. Biochemical and fatty acids composition were greatly influenced by both follicular dimension and phase of ovarian cycle. Biochemical composition and its variations were in agreement with previously study conducted in buffalo and other species. This is the first report of the fatty acids composition of buffalo follicular fluid. Twenty-two fatty acids were identified in follicular fluid; nine were saturated fatty acids, six monounsatured fatty acids and seven polyunsatured fatty acids. The most dominant fatty acids were linoleic acid, oleic acid, palmitic acid, stearic acid and arachidonic acid. All the identified fatty acids concentrations vary at least because of follicle dimension or phase, with the exception of γ-linoleic acid and arachidonic acid which concentrations remain stable in all classes.

Production by in vitro fertilization of vietnam swamp buffalo embryos

This study was undertaken in order to test the ability of replacement of the foetal bovine serum (FBS) supplemented with hormones by the buffalo follicular fluid (BFF) in the oocyte maturation medium and the effect of co-culture with oviduct cells on the subsequent embryo development in Vietnam swamp buffalo. 1400 cumulus enclosed oocytes (COC) were collected from the buffalo ovaries in slaughter-house in the way with minimize temperatural choc. COCs were randomtly cultured in three maturation medium varieties: TCM 199 supplemented with 10% FBS, 10 IU/ml eCG, 5 IU/ml hCG, 1 mg/ml estradiol (M1); TCM 199 with 20% buffalo follicular fluid (BFF) without hormones (M2) or TCM 199 with 20% BFF and the same level of eCG, hCG as M1 but 3 mg/ml estradiol (M3). The effect of oviduct cells was evaluated by the embryo co-culture in two medium varieties: synthetic oviduct fluid (SOF) and SOF with oviduct cells after the maturation in the medium M2 and IVF. No significal differences were observed in the maturation and the formation of morulas and blastocyts by using the routine maturation medium (M1) and TCM 199 supplemented with 20% BFF but the rate of formation of two-cell and more-cell embryos and the rate of total morula and blastocyts were clearly improved in the case of combined BFF and hormones (M3) (26.5% vs 16.3%, P &lt; 0.05). The co-culture with oviduct cells not only improved the percentage of total embryos developed into morula blastocyt stage but also increased the rate of blastocyt formation in comparision with the medium without cells (66.2% vs 44.4%, P &lt; 0.05). This first try of in vitro embryo production in the swamp buffalo indicated the comparable rate (approximately 20%) of blastocyts can be obtained from total using oocytes.

Expression of apoptosis related genes in buffalo embryos produced through in vitro fertilization and parthenogenetic activation

In present study immature oocytes were obtained from buffalo slaughterhouse ovaries and were subjected to in vitro maturation (IVM) in TCM-199 + 10% FBS + 5 µg/ml pFSH + 0.81 mM sodium pyruvate + 5% buffalo follicular fluid for 24 h in a CO2 incubator (5% CO2, 90–95% relative humidity) at 38.5°C. The maturer oocytes were used for IVF and the cleaved embryos were cultured for 8 days, while the parthenotes were produced with exposure of 7% ethanol, 6- dimethyl aminopurine and cultured for 8 days in mCR2aa medium. The relative abundance of BCL-XL transcripts in immature, matured oocytes; and embryos produced through IVF i.e. 2-cell, 4-cell, 8-16-cell, morula and blastocyst stage were 25.33±0.90, 12.67±1.20, 37.67±0.90, 30.67±0.30, 23.67±0.90, 18.33±0.90 and 27.00±1.20 respectively, while in parthenogenesis these values were 23.67±0.88, 13.67±1.20, 23.67±1.20, 22.34±0.88, 24.34±0.88, 33.67±0.88, 45.34±1.20 respectively. Relative abundance of BAX transcripts by IVF were 23.0±0.60, 0.33±0.10, 4.00±0.60, 5.00±0.60, 0.37±0.06, 13.0±0.66 and 56.7±0.90; and by parthenonenesis were 22.3±0.90, 0.13±0.03, 13.67±0.90, 14.0±0.60, 15.33±0.90, 64.67±2.20 and 55.0±2.10 respectively. In conclusion, expression pattern of apoptosis related genes revealed that incidence of apoptosis was significantly higher in IVF and parthenogenetically produced buffalo embryos at stages like immature oocytes, morula and blastocyst stage than the early cleavage stage embryos.

Changes in circulatory FSH of Barbari goats following treatment with high molecular weight inhibin isolated from buffalo follicular fluid

The objective of the present study was to isolate and purify high MW inhibin (∼129kDa) from buffalo ovarian follicular fluid (buFF) and to investigate its biological activity. Throughout the process of purification, the inhibin fractions were evaluated for bioactivity by a specific, sensitive and uniformly reproducible bioassay in mice. The final biological activity of this preparation was tested in normal cycling adult female Barbari goats. Eight animals, randomly divided into two groups, were synchronized for estrus by administering PGF2α twice at an interval of ten days. Following synchronization, the treatment group (n=4) received (i.m.) 0.4ml (240μg protein total dose) of purified inhibin (MW∼129kDa) of buFF in the morning at 08.00h for the four consecutive days of follicular phase, while the control group (n=4) received only saline (0.4ml). Blood samples were collected from jugular vein immediately before the first injection and subsequent collections were made daily in the afternoon until day 8 of the experiment including four days (0, 1, 2, & 3days) of the next cycle. FSH was assayed in all the samples by ELISA. The peripheral FSH concentration sharply declined from 1.854±0.137 to 0.979±0.02 u/l, 8h after the administration of inhibin on the first day. The value in controls was 2.004±0.132 u/l. For the duration of treatment of four consecutive days, the FSH level in experimental group remained significantly low (p<0.05) compared to control group. After cessation of treatment, the FSH level remained low on day 0 and 1 of the next cycle in the experimental and control animals. However, a significant rebound increase in plasma FSH levels occurred on day 2 & 3 (2.73±0.179 & 1.849±0.128u/l) only in the experimental group compared to control animals (P<0.05). This increment might be caused by the rebound surge of FSH from anterior pituitary which further corroborates the effect of inhibin in treated animals. In conclusion, the present study demonstrates that the high MW form of inhibin (∼129kDa) isolated from buFF has comparable biological activity as revealed by 31–32kDa inhibin from other species.

Impact of gonadotropin supplementation on the expression of germ cell marker genes (MATER, ZAR1, GDF9, and BMP15) during in vitro maturation of buffalo (Bubalus bubalis) oocyte

The present study was designed to investigate whether gonadotropins [follicle-stimulating hormone (FSH) and luteinizing hormone (LH)] and buffalo follicular fluid (bFF) supplementation in maturation medium influences the transcript abundance of germ cell marker genes [maternal antigen that embryos require (MATER), Zygote arrest 1 (ZAR1), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15)] mRNA in buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected from local abattoir, oocytes were aspirated from antral follicles (5-8 mm) and matured in vitro using two different maturation regimens, viz, group A: gonadotropin (FSH and LH) and group B: non-gonadotropin-supplemented maturation medium containing 20% buffalo follicular fluid (bFF). mRNA was isolated from immature (330) and in vitro matured oocytes from both the groups (A, 320; B, 340), and reverse transcribed using Moloney murine leukemia virus reverse transcriptase. Expression levels of MATER, ZAR1, GDF9, and BMP15 mRNA transcripts were analyzed in oocytes of both maturation groups as well as immature oocytes using real-time PCR. QPCR results showed that GDF9 and BMP15 transcripts were significantly (p<0.05) influenced with gonadotropins and bFF supplementation during in vitro maturation of buffalo oocyte; however, MATER and ZAR1 transcripts were not influenced with gonadotropins and bFF supplementation in vitro. These results indicated that the expression levels of MATER, ZAR1, GDF9, and BMP15 mRNA were varied differentially during in vitro maturation of buffalo oocyte and were found to be gonadotropins (FSH and LH) or bFF dependent for GDF9 and BMP15.

Presence of Nitric Oxide Synthase Immunoreactivity and mRNA in Buffalo (Bubalus bubalis) Oocytes and Embryos

This study examined the presence of immunoreactivity and mRNA for different nitric oxide synthase (NOS) isoforms in immature and in vitro matured oocytes and in embryos at two-, four- and eight-cell, and morula and blastocyst stages in buffalo. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to in vitro maturation in TCM-199 + 10% FBS + 5 μg/ml pFSH + 1 μg/ml estradiol-17β + 0.81 mm sodium pyruvate + 10% buffalo follicular fluid + 50 μg/ml gentamycin sulphate for 24 h in a CO(2) incubator (5% CO(2) in air) at 38.5°C. Following in vitro fertilization carried out by incubating them with 2-4 million spermatozoa/ml for 18 h, the presumed zygotes were cultured in mCR2aa medium containing 0.6% BSA and 10% FBS for up to 8 days post insemination. Immunofluorescence staining of NOS using antibodies that cross-reacted either with all the NOS isoforms i.e., universal (uNOS) or specifically with inducible (iNOS) or endothelial (eNOS) isoforms revealed that NOS was present in oocytes and embryos at all the stages examined. Examination of the semi-quantitative expression of NOS genes by RT-PCR revealed that the iNOS, eNOS and nNOS mRNA was present in the immature and mature oocytes and in all the embryonic stages examined. In conclusion, it was demonstrated in the present study that immunoreactivity and mRNA for different NOS isoforms was present in buffalo oocytes and pre-implantation stage embryos.

224 LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BUFFALO (BUBALUS BUBALIS) OOCYTES AND EMBRYOS

Ovarian nitric oxide (NO) and that produced within the oocytes and embryos have been reported to play important roles in oocyte meiotic maturation and embryo development. Production of NO is catalyzed by NO synthase (NOS), which exists in 3 isoforms, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible (iNOS) isoform. We have previously shown that low concentrations of NO stimulate and high concentrations inhibit embryo development, and that endogenous NO produced by iNOS is necessary for optimal embryo development in the buffalo. The present study was aimed at localizing different isoforms of NOS and examining their relative mRNA abundance in buffalo oocytes and embryos. Oocytes from slaughterhouse ovaries were subjected to in vitro maturation in 100-μL droplets (10 to 15 oocytes/droplet) of in vitro maturation medium (TCM-199 + 10% FBS + 5 μg mL–1 of pFSH + 1 μg mL–1 of oestradiol-17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 μg mL–1 of gentamicin) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. In vitro fertilization was carried out by incubating in vitro-matured oocytes with 2 to 4 million spermatozoa mL–1 for 18 h. The presumed zygotes were cultured on original beds of cumulus cells in in vitro culture medium (mCR2aa + 0.6% BSA + 10% FBS) for up to 8 days post-insemination. Immature and in vitro-matured oocytes and embryos at the 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stages were examined for the presence of NOS isoforms by indirect immunofluorescence staining using epifluorescence microscopy and RT-PCR. Each experiment was repeated in triplicate, and data were analysed using one-way ANOVA, after arcsine transformation of percentage values. Expression of all 3 NOS isoforms was detected inside the cytoplasm, in all the stages of oocytes and embryos examined, by both immunofluorescence and RT-PCR. Abundance of the iNOS transcript was significantly higher (P ≤ 0.01) in the morula and blastocyst stages compared with that in immature and in vitro-matured oocytes and in embryos at the 2-cell, 4-cell, and 8- to 16-cell stages, indicating that its expression was up-regulated at the 8- to 16-cell stage. The expression of eNOS was significantly higher (P ≤ 0.05) in the immature and mature oocytes and in 8- to 16-cell stage embryos, morulae, and blastocysts than in the early-cleavage embryos at the 2- and 4-cell stages, indicating that it was down-regulated after fertilization and was up-regulated again at the 8- to 16-cell stage. Abundance of the nNOS transcript was not significantly different among all the stages of oocytes and embryos examined. These results demonstrate that different NOS isoforms are expressed in a dynamic manner during embryonic development in the buffalo. The role of an increase in expression of iNOS and eNOS at the 8- to 16-cell stage, at which a developmental block occurs in this species, needs to be examined.

Bilateral follicular cysts in a water buffalo

The present short communication puts on record a case of bilateral, multiple follicular cysts in a water buffalo along with a detailed description of its ovarian biometry and follicular fluid composition. The ovarian weight and biometrical parameters were much higher than in normal cycling buffaloes. A total of three follicular cysts were observed, two on the right ovary and one on the left ovary, measuring 4.9, 3.0 and 2.6 cm yielding 21, 9 and 5 ml of follicular fluid, respectively. The cystic fluid was deep yellow in colour with a viscous consistency. The follicular fluid concentrations of glucose, total protein, cholesterol, acid phosphatase, calcium, phosphorus and progesterone in all the cysts were within the range reported previously in normal buffalo follicular fluid; however, the alkaline phosphatase concentration in cyst 1 and total bilirubin concentration in cysts 1 and 2 were higher than the values in normal follicular fluid. In contrast, the levels of urea nitrogen in cysts 1 and 3, and oestradiol in cyst 3 were lower than the normal values. All the three follicles had an oestradiol to progesterone ratio less than 1. The results of our study suggest that follicular cysts in buffalo are oestrogenically inactive and have an altered concentration of certain biochemical and hormonal constituents.

Multiple molecular forms of inhibin in buffalo (Bubalus bubalis) ovarian follicular fluid

Inhibin is a heterodimeric glycoprotein hormone involved in the regulation of FSH release from the anterior pituitary gland and it has been characterized from various animals. Although, multiple molecular forms of inhibin have been reported from different species, however, the molecular nature of inhibin has not been studied in buffaloes. In the present study, attempts were made to identify inhibin in buffalo ovarian follicular fluid. Buffalo ovaries were obtained from the local abattoir and follicular fluid was aspirated from surface follicle (with diameter> or =5mm). A combination of techniques (viz., gel filtration, SDS-PAGE, Western blot etc.) was employed for identification and isolation of inhibin(s). Inhibin bands were detected at 129 and 63 kDa by Western blot analysis in non-reducing conditions. In reduced SDS-PAGE, 63 kDa fraction produced a single band while 129 kDa fraction resolved into four components of 63, 43, 29 and 20 kDa. Out of them only 29, 63 and the native 129 kDa fractions produced bands on Western blot analysis. In total five fractions (63, 54, 39, 29, 25 kDa) were obtained by trypsin digestion of 129 kDa form. However, only 63 and 29 kDa fractions showed immunoreactivity. In this study, for the first time, we have identified two major forms of inhibin (129 and 63 kDa) with little proteolytic cleavage/processing of the large precursor in the buffalo follicular fluid.

Effect of exogenous administration of buffalo follicular fluid on follicular development, estrus response and luteal function in anoestrous goats ( Capra hircus)

The effect of buffalo follicular fluid (buFF) on follicular development, estrus response and luteal function was investigated in anoestrous does. Treatment with buFF (18 ml/doe) had no significant effect on the number of antral follicles of all class categories during the period of administration. However, after cessation of buFF treatment, the number of total antral follicles increased significantly with time ( P < 0.003) as well as due to the treatment × time interaction ( P < 0.02), without any influence on follicle size. Injection of buFF also caused a marked increase ( P < 0.049) with time in the number of medium-sized follicles at cessation. Approximately 60 and 20% of buFF-treated anoestrous does showed behavioural and silent estrus, respectively, compared to none in the control. The mean interval between cessation of buFF treatment to onset of oestrus and oestrus duration was 67.0 ± 18.5 and 17.0 ± 3.6 h, respectively. Corpus lutea size varied between 4.6 and 5.8 mm with an average diameter of 5.2 ± 0.3 mm. Only 33.3% of does showed serum progesterone levels above 1 ng/ml, while the remainder (66.7%) had below 0.5 ng/ml. Our results indicate that exogenous administration of buFF causes enhanced follicular activity following cessation of treatment, which results in behavioural oestrus and corpus luteum (CL) development in anoestrous does. CL development and its function is, however, inadequate in buFF-treated anoestrous does.

Effect of β-mercaptoethanol and buffalo follicular fluid on fertilization and subsequent embryonic development of water buffalo (Bubalus bubalis) oocytes derived from in vitro maturation

This study investigated effects of β-mercaptoethanol (β-ME) and buffalo follicular fluid (buFF) on IVF and embryonic development of water buffalo oocytes derived from IVM. In Experiment 1, β-ME (50µM and 100µM, treatments) was added in the media during IVM and IVC but not IVF. A highly significant difference was obtained in rate of blastocyst (9.26% Vs 26.72% or 27.04%, P<0.01), whereas no significant differences in rates of cleavage and hatched blastocyst between control group (0µM) and treatment groups (50µM and 100µM). Also, no significant differences (P>0.05) were observed in rates of cleavage, blastocyst and hatched blastocyst between two treatment groups, respectively. In Experiment 2, comparison with 10% FBS, significant and highly significant differences were recorded in rate of blastocyst in 5% FBS + 5% buFF (17.16% vs 27.64%, P<0.05)and 10% buFF (17.16% vs 32.98%, P<0.01), respectively. These results indicate that supplementation of β-ME in IVM and IVC and replacement of FBS with buFF in IVM media improve remarkably the development of water buffalo embryos derived from IVM-IVF to the blastocyst stage.

Effect of 30 kDa and above buffalo follicular fluid protein treatment and immunization on ovarian functions in goats ( Capra hircus)

Information on the use of buffalo follicular fluid (buFF) in modulation of ovarian functions in farm animals is scanty compared to other species. This is an attempt to investigate the effect of direct administration and active immunization of 30 kDa and above buFF proteins on ovarian functions in goats. Treatment of goats ( n = 6) with steroid free 30 kDa and above buFF protein fraction during late-luteal phase for 4 days (days 12 or 13 to days 15 or 16) of the natural cycle, delayed the onset of estrus by 24 h compared to control although the mean duration of estrus was unaffected. A 71% increase ( P = 0.06) in mean ovulation number was also observed following treatment. However, the population of large (≥5 mm diameter) follicle was not affected. The ovarian activity calculated as total of ovulation and large follicles increased (1.6 times) significantly ( P = 0.02) in treated animals. Active immunization of goats ( n = 5) against these proteins did not affect the onset and duration of estrus. Similarly, the ovulation rate, number of large follicles and the ovarian activity did not differ significantly between immunized and control groups. The study revealed that 30 kDa and above buffalo follicular fluid contains some factor(s) that cause delay in the onset of estrus in goats and increase the ovulation rate. Active immunization against these proteins in goat did not show any effect either on onset, duration of estrus or ovulation rate and large follicle population. Detailed study on these buffalo follicular fluid proteins may help to use them further for modulation of ovarian function in farm animals.

265EFFECT OF SPECIFIC GRAVITY OF CULTURE MEDIUM ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO

In buffalo the success rate of transferable quality embryo production through in vitro procedure is very low as compared to cattle. Sub optimal culture conditions and physical conditions such as specific gravity of the culture medium may lead to a reduced rate of transferable buffalo embryo production from the oocytes matured and fertilized in vitro (Palta &amp;amp; Chauhan,1998 Reprod. Fertil. Dev. 10, 379–391). This experiment was therefore conducted to find out the role of specific gravity of the IVC medium on the development rate of the buffalo embryos in vitro. Follicles of slaughter house ovaries were aspirated and the collected oocytes with cumulus-oocytes complexes (COCs) were cultured in TCM-199 medium supplemented with 10% fetal calf serum, 10% buffalo follicular fluid and 0.5μgmL−1 FSH in 5% CO2 incubator at 38.5°C. The matured oocytes were then inseminated with frozen-thawed buffalo semen suspended in BO medium. After 42h of post-inseminations the cleavage rates were evaluated. The 2–4 cell-cleaved eggs (Day 2 of post-insemination) were randomly divided and cultured for eight days in vitro in 1) modified synthetic fluid (mSOF)+0.8 %BSA (control), 2) mSOF+0.8 % BSA+gelatin (1mgmL−1) 3) mSOF+0.8% BSA+1mgmL−1 gelatin+10ngmL−1 epidermal growth factor (EGF). Supplementation of gelatin increased the specific gravity of the mSOF medium from 0.9658±0.009 to 1.0331±0.013 without any change in pH (7.4). The development of embryos to the 8–16 cell-stage on day 4 of in vitro culture were significantly higher (P&amp;lt;0.05) in mSOF+0.8% BSA+1mgmL−1 gelatin (81.8%; 27/33) than that in mSOF + 0.8% BSA (75.7%; 28/37) and mSOF+0.8% BSA+10ng/mL EGF (68.7%; 22/32). When these embryos were further cultured for another four days (Day 8), the development of transferable quality embryos (morula/blastocyst) was 42.4% (14/33), 48.7% (18/37) and 46.9% (15/32), respectively. Supplementation of gelatin increased the cleavage of eggs up to the 8–16 cell-stage embryo, but did not significantly enhance the rate of development to the morula/blastocyst stage in comparison to control and EGF-supplemented group. However, the percentage of transferable quality embryos was slightly lower in the gelatin-added group but not statistically significant than other groups. The study concluded that increase in specific gravity of the in vitro culture medium enhanced initial cleavage rate but did not have any role in transferable embryo production in buffalo.

INFLUENCE OF FOLLICLE SIZE ON QUALITY OF CUMULUS OOCYTE-COMPLEXES AND CONCENTRATION OF STEROIDS OF FOLLICULAR FLUID IN BUFFALO

The aim of the present study was to investigate the effect of follicle size on the buffalo cumulus oocyte complexes (COCs) morphology, chromatin configuration, level of apoptosis in cumulus cells, and concentration of progesterone and estradiol-17b in buffalo follicular fluid of different follicle size. Visible follicles were carefully isolated and classified according to their size into 3 categories 5 mm (5-8 mm). A total of 295 COCs were recovered from the previous three follicle categories; COCs were classified according to number and compactness of cumulus layers. Good quality compact COCs which used for IVM, which mostly obtained from follicles <3 and 3-5 (56-7%, 57.1) in comparison to 34.3% from large follicle. The chromatin configuration at the time of recovery in oocytes derived from 3-5 mm follicle respectively. There was a connection of cumulus morphology, chromatin configuration at the time of recovery and the follicle size.On the other hand no significant differences in maturation capacity (metaphase2)of COCs derived from different follicles diameter. Apoptosis of cumulus at time of recovery decreased with incre-asing the follicle size, but this difference was not significant (0.9, 0.6,0.2). After 24 hr. IVM, an increase in apoptosis was observed in cumulus cells from all follicle size with significant (P 5 mm follicles (24.4 Vs 4.7,3.5) Concentration of progesterone (ng/ml)in buffalo follicular fluid was not influenced by follicle size, while concentration of estradiol-17b (ng/ml) was significant higher in follicle 3-5, >5 mm (782 ± 67 and 788 ± 67 respectively) in comparison to follicular fluid from <3 mm follicles (682 ± 67).

Effect of Buffalo Follicular Fluid Alone and in Combination with PMSG and M199 on in vitro Buffalo Oocyte Maturation

Effect of Buffalo Follicular Fluid Alone and in Combination with PMSG and M199 on in vitro Buffalo Oocyte Maturation

R-001. Does buffalo follicular fluid enhance human sperm motility and vitality during activation in vitro?

R-001. Does buffalo follicular fluid enhance human sperm motility and vitality during activation in vitro?