In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis).

Cumulus-oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4 +/- 17.8 and 44.8 +/- 25.6, respectively. Addition of luteinizing hormone (LH) (5 micrograms ml-1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8 +/- 18.3), but follicle-stimulating hormone (FSH) (0.5 micrograms ml-1) and oestradiol (1 microgram ml-1) failed to synergize with LH (71.7 +/- 19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7 +/- 1.4 and 81.7 +/- 14.5, respectively; P less than 0.05). Frozen-thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1(-1) + 10 micrograms heparin showed a higher fertilization rate (29.8%) than those treated in Hepes-Talp and treated with 10 micrograms heparin ml-1 (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1-1 and 10 micrograms heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen-thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

Glutathione synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development

It was demonstrated that cysteamine supplementation during in vitro maturation (IVM) improves embryo development by increasing glutathione (GSH) synthesis in several species. An improved developmental competence of oocytes matured in the presence of cysteamine was also recorded in buffalo species. The purpose of this work was to investigate (1) if glutathione is de novo synthesized during in vitro maturation of buffalo oocytes, (2) if cysteamine improves buffalo embryo development via an increase in GSH synthesis, and (3) if the inhibition of glutathione synthesis by buthionine sulfoximide (BSO), in the presence or absence of cysteamine, affects subsequent embryo development and GSH synthesis.Cumulus-oocytes complexes (COCs), recovered from slaughtered animals, were matured in vitro in TCM199+10% fetal calf serum (FCS), 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17-beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 5mM BSO. Glutathione content was measured by high-performance liquid chromatography (HPLC) and fluorimetric analysis in immature oocytes and in oocytes matured in the different experimental conditions.In a second experiment, the mature oocytes were in vitro fertilized and cultured for 7 days in order to assess development to blastocysts (BLs). It was demonstrated that buffalo oocytes synthesize glutathione during in vitro maturation and that cysteamine increases glutathione synthesis. Furthermore, the promoting effects of cysteamine on embryo development and GSH synthesis were neutralized by buthionine sulfoximide. These results indicate that glutathione plays a critical role on buffalo embryo development.     

Ovarian follicular dynamics during the estrous cycle in buffalo (Bubalus bubalis)

The growth, selection, regression and ovulation of ovarian follicles was ultrasonically monitored in 30 Murrah buffalo throughout a spontaneous estrous cycle during the breeding season (autumn). Examinations revealed that follicular growth during the estrous cycle occurs in waves; the buffalo showed 1-wave (3.3%, n = 1), 2-wave (63.3%, n = 19) or 3-wave (33.3%, n = 10) follicular growth. The first wave began at 1.00, 1.16 +/-0.50 and 1.10 +/- 0.32 d in buffalo with 1, 2 and 3 waves, respectively (ovulation = Day 0). The second wave appeared at 10.83 +/- 1.09 and 9.30 +/- 1.25 d (P < 0.01) for the 2 and 3 wave cycle animals, respectively. The third wave started at 16.80 +/- 1.22 d. Structural persistence of the first dominant follicle was longer in the 2- than 3-wave cycles (20.67 +/- 1.18 vs 17.90 +/- 3.47 d ; P < 0.05). The duration of the growth and static phases of the first dominant follicle differed between the 2 and 3 wave cycles (P < 0.05), whereas there were no differences in linear growth rates (cm/d). Two and three wave cycles differed (P < 0.05) with respect to the maximum diameter of both the first dominant follicle (1.51 +/- 0.24 vs 1.33 +/- 0.18 cm) and the ovulatory follicles (1.55 +/- 0.16 vs 1.34 +/- 0.13 cm). No relationship was found between dominant follicle development and the presence of either a CL or a previous dominant follicle in either ovary. Two and three wave cycles also differed with respect to the mean length of intervals between ovulation (22.27 +/- 0.89 vs 24.50 +/- 1.88 d; P < 0.01) and the mean length of luteal phases (10.40 +/- 2.11 vs 12.66 +/- 2.91 d; P < 0.05). These results demonstrate that buffalo have estrous cycles with 1, 2 or 3 follicular waves; that 2-wave cycles are the most common; and that the number of waves in a cycle is associated with the luteal phase and with estrous cycle length.     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.     

Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods

The aim of this study was to evaluate the efficiency of the solid surface vitrification (SSV) and the cryoloop vitrification (CLV) methods to cryopreserve in vitro matured buffalo oocytes. Another objective of the work was to investigate whether the presence of cumulus cells affects the efficiency of oocyte vitrification in this species. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol, 5% polyvinyl-pyrrolidone and 0.4% trehalose and they were warmed in a 0.3M trehalose solution. In the CLV method, oocytes were vitrified in 16.5% ethylene glycol and 16.5% dimethyl sulfoxide and warmed in decreasing concentrations of sucrose. The oocytes that survived vitrification were fertilized and cultured in vitro up to the blastocyst stage. Although high survival rates were recorded in all groups, when the oocytes were vitrified by the CLV method in the absence of cumulus cells, the survival rate was significantly (P<0.05) lower. However, the CLV gave a significantly higher cleavage rate compared to the SSV with the denuded oocytes (45% versus 26%, respectively; P<0.05), whereas no differences were found between methods with the cumulus-enclosed oocytes (14% versus 15%, respectively). Blastocysts were produced for the first time from in vitro matured oocytes that were vitrified-warmed in buffalo. Nevertheless, vitrification significantly decreased blastocyst yield, regardless of both the method employed and the presence or absence of cumulus cells.     

Post-vitrification survival and in vitro maturation rate of buffalo (Bubalus bubalis) oocytes: effect of ethylene glycol concentration and exposure time

The present study was undertaken to investigate the effects of ethylene glycol concentration and time of exposure to equilibration solution on the post-thaw morphological appearance and the in vitro maturation rate of buffalo oocytes. Vitrification solution-I (VS-I) consisted of 4.5M ethylene glycol (EG), 3.4M dimethyl sulphoxide, 5. 56mM glucose, 0.33mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), whereas vitrification solution-II (VS-II) contained 3.5M EG, with other constituents at same concentrations as in VS-I. The equilibration solutions-I and II were prepared by 50% dilution (v/v) of VS-I and VS-II, respectively, in DPBS. Prior to vitrification, the cumulus-oocyte complexes (COCs) were exposed to equilibration solution-I or II for 1 or 3min at room temperature (25-30 degrees C). Groups of four to five oocytes were then placed in 15microl of respective vitrification solution, and immediately loaded into 0. 25ml French straws, each containing 150microl of 0.5M sucrose in DPBS. The straws were placed in liquid nitrogen (LN(2)) vapour for 2min, plunged and stored in LN(2) for at least 7 days. The straws were thawed by keeping in warm water at 28 degrees C for 20s, and the oocytes were equilibrated for 5min in 0.5M sucrose for one-step dilution. The percentage of oocytes found to be morphologically normal varied from 89 to 96% for the two equilibration solutions and the two exposure times. Among the damaged oocytes, cracking of zona pellucida was the abnormality observed most frequently. The nuclear maturation rate of oocytes equilibrated in equilibration solutions-I and II for 1 (28 and 24%, respectively) or 3min (32 and 33%, respectively) did not differ significantly. These results show that it is possible to cryopreserve buffalo oocytes by vitrification using a combination of 3.5M EG and 3.4M DMSO with an exposure time of 3min.     

Changes in biochemical composition of follicular fluid during reproductive acyclicity in water buffalo (Bubalus bubalis)

This study describes the changes in biochemical composition of follicular fluid during reproductive acyclicity in buffalo. A total of 73 pairs of ovaries collected from 26 reproductively acyclic and 47 reproductively cyclic buffaloes were used in the investigation. Ovarian follicles were classified into small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized categories depending upon their diameter. Follicular fluid was aspirated, processed and assayed for glucose, cholesterol, total protein, acid phosphatase and alkaline phosphatase. Glucose concentration was lesser in reproductively acyclic compared to cyclic buffaloes (19.3 ± 2.59 mg/dl compared to 32.6 ± 2.60 mg/dl; P<0.05), mainly due to difference in concentration between small sized follicles (12.4 ± 2.59 mg/dl compared to 28.0 ± 3.32 mg/dl; P<0.05). Cholesterol concentration was also lesser in reproductively acyclic compared to cyclic buffaloes (32.2 ± 2.14 mg/dl compared to 35.5 ± 2.16 mg/dl; P<0.05) and this was related to the lesser concentration found in large follicles (13.8 ± 3.45 mg/dl compared to 37.2 ± 4.10mg/dl; P<0.001). Total protein and acid phosphatase levels were not affected by either the reproductive cyclicity status or the follicular size (4.9 ± 1.07 g/dl to 6.0 ± 0.28 g/dl and 1.2 ± 0.17 U/dl to 2.5 ± 1.22 U/dl, respectively). An increased alkaline phosphatase activity was, however, observed in reproductively acyclic compared to cyclic buffaloes (27.5 ± 3.08 U/dl compared to 14.0 ± 1.09 U/dl; P<0.0001). In conclusion, results of the present study indicate an alteration in the biochemical composition of follicular fluid during reproductive acyclicity in buffalo. The findings provide further support to the notion that poor nutrition is an important factor triggering reproductive acyclicity in buffalo.     

Identification of Sarcocystis hominis-like (Protozoa: Sarcocystidae) cyst in water buffalo (Bubalus bubalis) based on 18S rRNA gene sequences

DNA templates were extracted from isolates of Sarcocystis hominis-like cysts collected from cattle and water buffalo, as well as from Sarcocystis fusiformis cysts and Sarcocystis suihominis cysts. The 18S rRNA genes were amplified using DNA from a single cyst as the templates. Approximately 1,367-1,440 bp sequences were obtained. The sequence difference in isolates of Sarcocystis hominis-like cysts from water buffaloes, and isolates of S. hominis cysts from cattle were very low, only about 0.1%, much lower than the lowest value (1.7%) among different species. Combined with their morphological structure, these sequence data indicate that the 4 isolates from cattle and water buffalo might be the same species, i.e., S. hominis, suggesting that both cattle and water buffalo may serve as the intermediate hosts for this parasite. Apparently, this is the first report using a single cyst to do such work and is a useful way to distinguish the Sarcocystis cyst in an intermediate host that may be simultaneously infected by several different Sarcocystis species.     

In vitro culture of buffalo (Bubalus bubalis) preantral follicles

Growth of buffalo preantral follicles in culture was studied to investigate the effect of size of preantral follicles, individual or group culture, long-term culture of preantral follicles for (40 days), addition of human follicle stimulating hormone (FSH), insulin-transferrin-selenium (ITS), growth factors (epidermal growth factor (EGF), fibroblast growth factor (FGF), vaso active intestinal polypeptide (VIP) in culture media, and substitution of pregnant mare serum gonadotrophin (PMSG) for FSH as gonadotrophin source in culture media. Preantral follicles were isolated mechanically from ovaries of matured, nonpregnant slaughtered buffaloes and cultured in droplets of culture media under mineral oil in a 35 mm petri dish in a CO2 incubator (38-39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 15 days. Preantral follicle isolation and washing medium consisted of Minimum Essential Medium (MEM) supplemented with steer serum (10%), glutamine (2 mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM) and gentamycin (50 microg/ml), respectively. In Experiment 1, we placed isolated preantral follicles individually or in groups of 2-4 preantral follicles in 30 or 50 microl droplets, respectively, using two culture media: washing media and washing media + ITS (1%) + FSH (0.05 IU/ml), respectively. In Experiment 2, we grouped isolated preantral follicles were grouped into six different size classes: < or = 36, 37-54, 55-72, 73-90, 90-108 and > or = 109 microm. We cultured groups of 2-4 preantral follicles in washing media + ITS (1A) + FSH (0.05 IU/ml) in a CO2 incubator for 15 days. In Experiment 3, we allocated groups of 2-4 preantral follicles to 10 treatments: (1) only washing media, (2) washing media + FSH (0.05 IU/ml), (3) washing media + ITS (17%), (4) washing media + ITS (1%) + FSH (50 IU/ml), (5) washing media + ITS (1%) + EGF (50 ng/ml), (6) washing media + ITS (1%) + FSH (0.05 IU/ml) + EGF (50 ng/ml), (7) washing media + ITS (1%) + FGF (50 ng/ml), (8) washing media + ITS (1%) + FSH (0.05 IU/ml) + FGF (50 ng/ml), (9) washing media + ITS (1%) + VIP (50 ng/ml), and (10) washing media + ITS (1%) + FSH (0.05 IU/ml) + VIP (50 ng/ml). In Experiment 4, based on the results of Experiment 3, we incubated preantral follicles from those treatments showing significantly (P < 0.05) higher growth up to 40 days. In Experiment 5, we allocated groups of 2-4 preantral follicles to two treatments: (1) washing media + PMSG (50 IU/ml), and (2) washing media + ITS (1%) + PMSG (50 IU/ml) and cultured in a CO2 incubator for 15 days. The results indicated that the preantral follicles cultured in groups had a higher growth rate (P < 0.05) than those cultured as individuals. ITS, FSH, PMSG and growth factors significantly (P < 0.05) promoted the growth of the preantral follicles. Following 40 days of culture, follicular architecture was preserved in nearly 17% of the follicles though there was no antrum formation. The growth rate of preantral follicles was lower in buffalo than in cattle.     

Characterization of PRLR and PPARGC1A genes in buffalo (Bubalus bubalis)

More than 40 million households in India depend at least partially on livestock production. Buffaloes are one of the major milk producers in India. The prolactin receptor (PRLR) gene and peroxisome proliferators activated receptor-γ coactivator 1-alpha (PPARGC1A) gene are reportedly associated with milk protein and milk fat yields in Bos taurus. In this study, we sequenced the PRLR and PPARGC1A genes in the water buffalo Bubalus bubalis. The PRLR and PPARGC1A genes coded for 581 and 819 amino acids, respectively. The B. bubalis PRLR gene differed from the corresponding Bos taurus at 21 positions and four differences with an additional arginine at position 620 in the PPARGC1A gene were found in the amino acid sequence. All of the changes were confirmed by cDNA sequencing. Twelve buffalo-specific single nucleotide polymorphisms (SNPs) were identified in both genes, with five of them being non-synonymous.     

Effects of bull and heparin and sperm concentrations on in vitro fertilization of buffalo (Bubalus bubalis ) oocytes matured in vitro

A study was undertaken to assess the ability of spermatozoa from 6 buffalo bulls, at different levels of heparin and sperm concentrations, to achieve an acceptable level of fertilization in vitro. Frozen-thawed spermatozoa, 3 dosages of heparin (0, 10 and 100 ug/ml) in the presence and absence of penicillamine, hypotaurine and epinephrine (PHE), and 4 sperm concentrations (1 x 10(6), 2 x 10(6), 3 x 10(6) and 4 x 10(6) /ml) were studied using 3202 buffalo oocytes. The mean proportions of fertilized oocytes in the group treated with 10 ug/ml of heparin were significantly higher (P<0.05) with the semen of Bulls A, B and C (44.7 to 64.3%) than in medium devoid of heparin. An increase in the dosage of heparin from 10 ug/ml to 100 ug/ml reduced the overall fertilization rate. However, optimal fertilization (30.9%) at 100 ug/ml heparin was observed for semen from Bull D. Bulls E and F yielded the lowest fertilization rate (9.6 and 14.2%, respectively) at the above mentioned heparin dosage. Analysis of sperm density revealed that a concentration of 2 x 10(6) spermatozoa yielded optimal fertilization rates in vitro. Higher sperm concentrations (3 x 10(6) or 4 x 10(6)) resulted in higher oocyte penetration rates but gave rise to polyspermy.     

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.     

Effect of cryoprotectants and their concentration on in vitro development of vitrified-warmed immature oocytes in buffalo (Bubalus bubalis)

Experiments were conducted to study the effect of cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (PROH), and glycerol at different concentrations (3.5, 4, 5, 6, and 7 M each with 0.5 M sucrose and 0.4% BSA in DPBS) on survival, in vitro maturation, in vitro fertilization, and post-fertilization development of vitrified-thawed immature buffalo oocytes. The COCs were harvested from the ovaries by aspirating the visible follicles. The recovery of post-thaw morphologically normal oocytes was lower in 3.5 and 4 M DMSO, EG, and PROH compared to 5, 6, and 7 M. In all the concentrations of glycerol, an overall lower numbers of oocytes recovered were normal compared to other cryoprotectants. Less number of oocytes reached metaphase-II (M-II) stage from the oocytes cryopreserved in any of the concentrations of DMSO, EG, PROH, and glycerol compared to fresh oocytes. Among the vitrified groups, highest maturation was obtained in 7 M solutions of all the cryoprotectants. The cleavage rates of oocytes vitrified in different concentrations of DMSO, EG, PROH, and glycerol were lower than that of the fresh oocytes. The cleavage rates were higher in oocytes cryopreserved in 6 and 7 M DMSO, EG, PROH, and glycerol compared with oocytes cryopreserved in other concentrations. However, the percentage of morula and blastocyst formation from the cleaved embryos did not vary in fresh oocytes and vitrified oocytes. In conclusion, this report describes the first successful production of buffalo blastocysts from immature oocytes cryopreserved by vitrification.     

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.     

Alterations in follicular fluid estradiol, progesterone and insulin concentrations during ovarian acyclicity in water buffalo (Bubalus bubalis)

Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized follicles. Estradiol concentration was lower (P<0.0001) in acyclic (1.4 ± 0.09 ng/ml) than in cyclic (3.3 ± 0.18 ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P<0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4 ± 0.16 ng/ml, 2.8 ± 0.29 ng/ml and 3.5 ± 0.41 ng/ml in small, medium and large follicles, respectively. A higher (P<0.001) progesterone concentration was recorded in acyclic (24.3 ± 2.61 ng/ml) compared to the cyclic (7.6 ± 0.79 ng/ml) group. Furthermore, acyclic buffaloes had a lower (P<0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2 ± 1.55 μIU/ml versus 25.9 ± 2.78 μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes.     

Effect of slow freezing on morphology and developmental competence of buffalo (Bubalus bubalis) immature oocytes

The present study was conducted to evaluate the effects of three cryoprotectants, dimethyl sulphoxide (DMSO), ethylene glycol (EG) and 1,2-propanediol (PROH), each used at two concentrations (1.0 and 1.5 M) on the morphology, maturation rate and developmental capacity of usable quality immature buffalo oocytes subjected to slow freezing. The addition of the cryoprotectant before freezing and its dilution after thawing were carried out in a two- (for 1.0 M) or three-step manner (for 1.5 M). The incidence of damage was found to be significantly higher (P<0.05) with the lower concentration of 1.0 M, compared to that with 1.5 M for all the three cryoprotectants examined. The proportion of immature oocytes recovered in a morphologically normal state was significantly higher (P<0.05) for DMSO than those for EG or PROH at both 1.0 and 1.5 M concentrations. Among the six combinations evaluated, that of DMSO at 1.5 M concentration was found to be superior to others. Irrespective of the type or concentration of the cryoprotectant, partial or complete loss of the cumulus mass was the most prevalent damage. Following in vitro maturation, the nuclear maturation rate was significantly higher (P<0.05) for DMSO than those for EG or PROH at both 1.0 and 1.5 M concentrations. When the in vitro matured oocytes were subjected to in vitro fertilization after slow freezing, using 1.5 M DMSO as cryoprotectant, 4.5% and 0.6% of them were able to develop to morulae and blastocysts, respectively, on Day 9 post insemination, compared to 19.2% and 10.6%, respectively, for the controls. In conclusion, DMSO was more effective than EG or PROH for the slow freezing of immature buffalo oocytes and blastocysts could be produced from immature buffalo oocytes subjected to slow freezing in 1.5 M DMSO.