Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).     

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.     

Storage of buffalo (Bubalus bubalis) semen

Characteristics of buffalo semen, diluents used for liquid storage, aspects involved in freezing and thawing of semen are reviewed, and fertility results after artificial insemination (AI) with frozen-thawed semen are given.     

Embryo transfer in water buffalo (Bubalus bubalis)

A normal, live 35-kg water buffalo bull calf was born 300 days after it was nonsurgically collected as a 7-day blastocyst from a water buffalo donor and transferred nonsurgically to an unrelated water buffalo recipient. The development of estrus synchronization, superovulation and estrus detection methods in water buffalo are described.     

Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The objective of this study was to identify a Y chromosome-specific sequence in water buffalo and to establish an efficient procedure for embryo sexing by LAMP. The homologues of a Y chromosome-specific sequence, bovine repeat Y-associated.2, in swamp and river buffalo were cloned, and designated swamp buffalo repeat Y-associated.2 and river buffalo repeat Y-associated.2, respectively. Sexing by LAMP was performed using primers for swamp buffalo repeat Y-associated.2. A 12S rRNA was also amplified by LAMP as a control reaction in both male and female. The minimal amount of the template DNA required for LAMP appeared to be 0.1-10 pg. The sensitivity was further examined using swamp buffalo fibroblasts as templates. When fibroblasts were lysed with NaOH, the minimal cell number required for detection of both male-specific and male-female common DNA appeared to be two cells, whereas correct determination of sex could not be achieved using fibroblasts lysed by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies nuclear transfer embryos. The sex determined by LAMP for blastomeres corresponded with the sex of nuclear donor cells in analyses using four or five blastomeres as templates. The LAMP reaction required only about 45 min, and the total time for embryo sexing, including DNA extraction, was about 1 h. In conclusion, the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos.     

Rate of transport and development of preimplantation embryo in the superovulated buffalo (Bubalus bubalis)

This study was designed to ascertain the rate of transport and development of preimplantation embryo in the superovulated buffalo in order to determine the optimum time for their nonsurgical collection. Eighteen Murrah-type buffalo were superovulated with 600 mg NIH-FSH-P1. Luteolysis was induced by administration of PGF2 alpha at 72 (PG + 72) and 84 h (PG + 84) after initiating gonadotrophin treatment and fixed-time AI was done beginning at 36 h post PG + 72 administration and at 12-h intervals thereafter, upto 72 h. Six control buffalo received treatment similar to experimental group except that in place of FSH they received normal saline. For embryo collection, experimental animals were humanely killed at 6-h intervals corresponding to 156 (n = 2), 162 (n = 2), 168 (n = 2), 174 (n = 3), 180 (n = 3), 186 (n = 3) and 192 h(n = 3) after PG + 72 treatment, whereas the control animals were humanely killed at 156 (n = 2), 174 (n = 2) and 192 h (n = 2). Superovulated buffalo had higher number of ovulations than untreated controls (8.78 +/- 5.00 vs 0.67 +/- 0.51) and total ova/embryos recovered was 4.11 +/- 2.46 and 0.67 +/- 0.51, respectively. The high estradiol-17 beta (E2) levels with its prolonged rise may, by leading to reverse peristalsis in the oviduct with a consequent loss of some embryos in the peritoneal cavity, be one of the reasons for our inability to recover nearly 84/158 ova/embryos in the superovulated buffalo. In superovulated animals, nearly all the ova/embryos reached the uterus between 168 and 174 h post PG + 72 treatment or about 134 h (circa 5.5 d) after the onset of superovulatory estrus, suggesting that the ideal time for non-surgical embryo collection in the buffalo is between Days 7 to 8 after PG + 72 treatment or Days 5.5 to 6.0 of the superovulated cycle (estrus = Day 0). Embryo development of superovulated buffalo showed considerable variation as various stages of embryos (8 cell to expanded blastocyst) were recovered from the same donor buffalo, and the rate of development appeared to be 24 to 36 h faster than in cattle.     

Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley-Liss, Inc.     

Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.     

Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems

Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p < 0.05) blastocyst rates than Well of the Wells (WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.     

Total lipids and phospholipids in buffalo semen (Bubalus bubalis)

Spermatozoal and seminal plasma concentrations of total lipids from 50 ejaculates and phospholipids and their fractions from 30 ejaculates were quantified in the semen of five Murrah buffalo bulls. Sperm lipid content ranged from 0.93 to 1.72 mg/10(9) cells with an overall average 1.32 +/- 0.03 mg/10(9) cells. Its concentration in seminal plasma varied from 1.39 to 2.22 mg/ml with overall average of 1.75 +/- 0.03 mg/ml. Spermatozoal total phospholipid content ranged from 0.44 to 0.94 mg/10(9) cells with overall mean being 0.64 +/- 0.02 mg/10(9) cells. The corresponding values for seminal plasma were 0.53 and 0.88 mg/ml with an overall mean of 0.69 +/- 0.02 mg/ml. Phosphatidyl choline constituted the major fraction both in the spermatozoa and and seminal plasma.     

The serum lipoprotein pattern of water buffalo (Bubalus bubalis)

1. The serum lipoprotein pattern of water buffalo was studied by means of electrophoresis and the lipoproteins were isolated by ultracentrifugation on the basis of their hydrated density. 2. High density lipoproteins (HDL) showed a higher level of cholesterol than did the other lipoproteins. Moreover, the level of phospholipids was higher in HDL than in very low density lipoproteins (VLDL). 3. The buffalo B100 apoprotein was similar to that of man and rat. Three apoproteins similar to human apo E, apo AI and AII were found in buffalo HDL, buffalo VLDL contained essentially apo B protein.     

Development of swamp buffalo (Bubalus bubalis) embryos after parthenogenetic activation and nuclear transfer using serum fed or starved fetal fibroblasts

The knowledge of oocyte activation and somatic cell nuclear transfer in the swamp buffalo (Buballus bubalis) is extremely rare. The objectives of this study were the following: (1) to investigate the various activation treatments on the parthenogenetic development of buffalo oocytes, (2) to examine the events of nuclear remodeling and in the in vitro development of cloned buffalo embryos reconstructed with serum fed or starved fetal fibroblasts, and (3) to investigate the in vivo development of cloned embryos derived from serum fed or starved cells after transfer into the recipients. The rates of cleavage and blastocyst development were found to be significantly higher (P < 0.05) when the oocytes were activated by the combination treatment of calcium ionophore (A23187) or ethanol followed by 6-DMAP than those activated by electrical pulses and 6-DMAP or other single treatments. Flow cytometric analysis revealed that the percentage in the G0/G1 phase in serum starved cells was significantly (P < 0.05) higher than that in serum fed cells (88.8 +/- 6.2 vs. 68.2 +/- 2.6). At 1 h post fusion (hpf), most of the transferred nuclei (71%) from serum fed cells did not change in size, and the nuclear envelope remained intact, whereas 29% underwent NEBD and PCC. When serum starved cells were used, 83% of the transferred nuclei underwent NEBD and PCC whereas 17% remained intact. The nuclear swelling and pronucleus (PN) formation were observed at 2-4 and 12 h post activation (hpa), respectively. The remodeled nuclei underwent mitotic division and developed to the 2-cell stage within 18-24 hpa. Fifty-five percent of oocytes reconstructed with serum fed cells were 2PN and 45% were 1PN, whereas 79% of the embryos reconstructed from starved cells were 1PN and 21% were 2PN. The percentage of blastocyst development of the embryos derived from starved cells was higher than that from the serum fed cells (35% vs. 21%, P < 0.05). Pregnancy was detected after the transfer of cloned blastocysts into the recipients but no recipients supported the development to term. The results of this work can be used to establish effective activation protocols for buffalo oocytes which can be used during nuclear transfer experiments.     

Successful nonsurgical embryo transfer in buffalo (Bubalus bubalis ) in Bulgaria

Forty-one Day 5.0 to Day 5.5 embryos and one unfertilized ovum were recovered nonsurgically from 24 superovulated, parous buffalo (Bubalus bubalis ) and transferred nonsurgically to 28 synchronized recipients by a team of Bulgarian and American scientists. Five pregnancies were established and four live buffalo calves were born at the end of normal gestation periods.