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.     

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.     

The autonomous innervation of the buffalo (Bubalus bubalis) testis. An immunohistochemical study

The innervation pattern in the buffalo testis was determined by using histochemical and immunohistochemical methods. Nerves were concentrated in the tunica albuginea and septula testis, and did not show an uniform distribution. The tunica albuginea at the lateral and medial sides and at the free border of the testis is most densely innervated than at the epididymal border. At the cranial pole thick nerve bundles were observed between albugineal vessels and muscle bundles. Rare parenchymal nerves were found in perivascular position between seminiferous tubules and their occurrence is confined to lobules at the cranial and caudal testicular poles. An intense NPY immunoreactivity occurred in nerve bundles and in solitary varicose fibres. Nerves were concentrated in the tunica albuginea at the lateral and medial side and at the free border of the testis, and in the lobules at the cranial and caudal testicular poles. Sub P immunoreactivity was occasionally detected in some thicker nerve bundles and solitary fibers, in the tunica albuginea and in the wall of blood vessels, showing a similar distribution but less intensity and density than NPY immunoreactivity. TH immunoreactivity stained nerve fibers in the buffalo testis with a distribution pattern similar to that obtained with general neuronal markers. The histochemical reaction for AchE was negative, so cholinergic fibers cannot be detected in the buffalo testis. The histochemical NADPHd reaction stained rare nitrergic nerve bundles and solitary fibers. The majority of NADPHd activity was confined to the vascular endothelium, and rarely to the interstitial Leydig cells, whereas the Sertoli and germ cells did not show any reaction.     

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.     

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β(1) domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.     

Experimental Sarcocystis hominis infection in a water buffalo (Bubalus bubalis)

A water buffalo (Bubalus bubalis) was fed 5.0 x 10(5) Sarcocystis hominis sporocysts from a human volunteer who had ingested S. hominis cysts from naturally infected cattle. A necropsy was performed on the buffalo 119 days after inoculation, and a large number of microscopic sarcocysts (approximately 5,000/g) were found in skeletal muscles. Ultrastructurally, the sarcocyst wall from buffalo muscles has upright villar protrusions measuring about 5.6 x 0.8 microm with numerous microtubules that run from the base to the apex. Sarcocysts from this buffalo were infective to 2 human volunteers, confirming their identity as S. hominis. Therefore, we believe that buffaloes can act experimentally as the intermediate host for S. hominis.     

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.     

Genetic variation within and relationships among populations of Asian water buffalo (Bubalus bubalis)

Genetic variation at 53 protein-coding loci (25 polymorphic) was analysed for 17 water buffalo populations – 12 swamp, three Lankan and two of the Murrah breed (river type), to determine the magnitude of genetic differentiation and the genetic relationships among the populations. In accord with previous cytological studies, the Lankan buffalo clearly are river type. Significant deviations from Hardy–Weinberg equilibrium were shown for a number of locus–population combinations, with all populations but one showing significant heterogeneity in these deviations among loci. By contrast, heterogeneity among populations for each locus was much less, indicating locus-specific deviations, which suggest selection affecting allele frequencies at some loci. There was significant genetic differentiation among populations of both the swamp and river types. The differentiation among the swamp populations may reflect the geography of south-east Asia and the presumed spread of the swamp buffalo through this region. Phylogenies derived from pairwise genetic distance estimates show the clear separation of swamp and river types, but the topology of the swamp populations shows rather poor consistency with their geographic locations. For at least one population (Australia), it is clear that bottleneck effects have distorted the phylogenetic topology. Average genetic distances for both the swamp and river types, as compared with previous studies of livestock breeds, show that the genetic differentiation of each of these sets of populations is of the same order of magnitude as that among well-recognized and established breeds of other species.     

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.     

Identification of X- and Y-chromosome bearing buffalo (Bubalus bubalis) sperm

The aim of this study was to identify the difference in DNA content characterizing the X- and Y-chromosome bearing sperm of buffalo. Sperm from six Murrah buffaloes and six Nili-Ravi buffaloes were collected and stained with Hoechst 33342 followed by flow cytometry analysis of the DNA content. Two symmetrical, separate but overlapping peaks presumed to be X- and Y-chromosome bearing sperm were detected. The difference in fluorescence intensity, which related to the DNA content, between the X- and Y-sperm was 3.59+/-0.11% for Murrah buffalo and 3.55+/-0.14% for Nili-Ravi buffalo, respectively. Significant differences were observed among males within each breed, but there were no differences between the averages of the two breeds. The results indicate that flow cytometric sorting of X- and Y-sperm of buffalo is feasible.     

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.     

The Sertoli cell of the water buffalo (Bubalus bubalis) during the spermatogenic cycle

Summary Ultrastructural features and morphometric evaluations of buffalo Sertoli cells are reported for the six phases of the spermatogenic cycle. The phases of the tubular seminiferous epithelium are identified according to characteristic cellular associations with completed spermiation as demarcation between two cycles. Average tubular diameter (245 m) and epithelial height (61 m) do not vary significantly during the cycle. The relative Sertoli cell volume in the seminiferous epithelium varies between 30% (phase 4) and 39% (phase 8). The calculated volume of a single Sertoli cell increases from a nadir of 7118 m³ in phase 3 abruptly to a maximum of 8968 m³ in phase 4 and is then gradually reduced during the following phases. The Sertoli cell surface area shows a similar trend: it amounts to 11105 m² in phase 3 and to 14260 m² in phase 4. The contact area of the Sertoli cell with adjacent cells and structures is subject to characteristic changes; from the expansion of basal Sertoli-Sertoli contacts it is concluded that the blood-testis barrier in the buffalo is particularly tight during phases 8, 1 and 2. The irregularly contoured nucleus contains a vesicular nucleolus, has a calculated volume from 465 m³ to 543 m³ and occupies 5 to 7% of the cell. Volume percentages of mitochondria (4%), Golgi apparatus and lysosomal bodies are rather constant during the cycle. Whorls and orderly arranged aggregates of the smooth endoplasmic reticulum occur in basal location as well as in close association with elongating spermatids. Smooth ER is the organelle that exhibits the most prominent changes during the Sertoli cell cycle: it occupies 5.79% in phase 3 and 20.9% in phase 4 of the total cellular volume. Phagocytosis of residual bodies is insignificant in this species and a lipid cycle is absent in buffalo Sertoli cells.     

Independent maternal origin of Chinese swamp buffalo (Bubalus bubalis)

To obtain more knowledge on the origin and genetic diversity of the swamp buffalo (Bubalus bubalis) in China, the complete mitochondrial D-loop sequences of 119 samples representing seven native types were compared. Two mitochondrial DNA (mtDNA) lineages (lineages A and B) were determined for the Chinese swamp buffalo. Examination of the diversity patterns suggest that lineage A has undergone a population expansion event. Divergence of lineages A and B was estimated at 18,000 years ago. Combined analyses of mtDNA sequences from Chinese, Indian, Brazilian/Italian and Southeast Asian/Australian buffalo samples showed independent domestication events in the swamp buffalo from China and the river buffalo from the India subcontinent. The spread of swamp and river buffalo from China and India respectively to mainland Southeast Asia suggests that Southeast Asia is a hybrid zone for buffalo. Our data support the hypothesis of the evolution of domesticated swamp and river buffalo from ancestral swamp-like animals. These ancestral animals were extensively distributed across mainland Asia and most likely are represented today by the wild Asian buffalo (Bubalus arnee).     

Mannose-binding lectin haplotypes influence Brucella abortus infection in the water buffalo (Bubalus bubalis)

A case-control study established that the haplotype pair HYA/HYA at the MBL (mannose binding lectin) locus of water buffalo is associated with resistance to Brucella abortus infection (P < 10⁻⁷) and the haplotype pairs LYD/LYD with susceptibility to the same pathogen (P < 10⁻⁷). The subjects included in the present study were tested twice—at a 1-month interval—for the presence of anti-B. abortus antibodies in the serum by agglutination, complement fixation and flow cytometry. Cases (335 subjects) included animals consistently positive to all these tests; controls (335 subjects) comprised animals exposed yet negative by the same tests. The serum from genetically resistant subjects displayed in vitro significantly higher antibacterial activity compared to the serum from genetically susceptible subjects, lending biological significance to the results from the association study. Inhibition of the antibacterial activity following heat treatment of the serum, addition of specific MBL inhibitors (EDTA, mannose, N-acetyl-d-glucosamine) or anti-human MBL antiserum provide convincing evidence that the antibacterial activity present in the serum results from the interaction between MBL and B. abortus. A replication study (comprising 100 cases and 100 controls) confirmed the results from the original study.