Integrated analysis of phosphoproteome and ubiquitylome in epididymal sperm of buffalo (Bubalus bubalis)

In mammals, sperm need to mature in the epididymis to gain fertilization competency. However, the molecular mechanism underlying buffalo sperm maturation remains elusive. Exploring sperm physiology at the posttranslational modification (PTM) level could help to develop our understanding of these mechanisms. Protein phosphorylation and ubiquitination are major PTMs in the regulation of many biological processes. In the present study, to our knowledge, we report the first phosphoproteome and ubiquitylome of sperm collected from the caput, corpus, and cauda segments of the epididymis using liquid chromatography–mass spectrometry combined with affinity purification. In total, 647 phosphorylation sites in 294 proteins and 1063 ubiquitination sites in 446 proteins were characterized. Some of these proteins were associated with cellular developmental processes and energy metabolic pathways. Interestingly, 84 proteins were both phosphorylated and ubiquitinated, simultaneously. Some of these proteins were involved in, for example, spermatogenesis, reproduction, and spermatid development. Taken together, these data provide a theoretical basis for further functional analysis of phosphorylation and ubiquitination in epididymal sperm of buffalo and other mammals, and serve as an important resource for exploring the physiological mechanism underlying sperm maturation.     

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).     

Treatment with chemical delipidation forskolin prior to cryopreservation improves the survival rates of swamp buffalo (Bubalus bubalis) and bovine (Bos indicus) in vitro produced embryos

The cryopreservation of embryos is a technology developed for long-term genetic preservation. However, high sensitivity to low temperatures due to a large number of intracellular lipids within ruminant embryos compromises the success of this technique. The aim of this study was to examine the effects of using of lipolytic chemical agent forskolin, during in vitro producing of buffalo and bovine embryos on lipid contents, cryotolerance and subsequent developmental competence of these embryos. Buffalo and bovine oocytes were collected by the aspiration technique from follicles and submitted for in vitro fertilisation; the embryos were later divided into four experiments. Experiment 1, buffalo and bovine embryos were pre-treated in the presence and absence of 10 μM forskolin for 24 h. Lipid contents were determined by Nile red staining and confocal microscopy. We found that 10 μM forskolin was capable to reduce lipid contents within developing embryos in both of species (P < 0.01). Lipid contents within Day 2 embryos exhibited greater fluorescence intensity than did Day 7 embryos in both animal species. The purpose of Experiment 2 was to investigate the adverse effects of 10 μM forskolin on embryo development. In Experiments 3 and 4, Day 2 (4- to 8-cell stage) and Day 7 (blastocyst stage) embryos were pre-treated with 10 μM forskolin for 24 h and further cryopreserved with a controlled-rate freezing technique. The successful cryopreservation was determined by post-thawed embryonic development in vitro. The results showed that the blastocyst rate of the 4–8 cell stage in the forskolin-treated group had increased in both species, while the hatching and hatched blastocyst rates of forskolin-treated day 7 bovine embryos were significantly higher than those of the non-treated group (52.1% vs. 39.4%; P < 0.05). However, delipidation with forskolin did not affect the developmental rate of the day 7 buffalo embryos (P = 0.73). Our studies showed that delipidation by forskolin treatment increased the survival rate of cryopreservation in buffalo and bovine in vitro produced embryos.     

A set of polymorphic DNA microsatellites useful in swamp and river buffalo (Bubalus bubalis)

DNA microsatellites have found widespread application in gene mapping, pedigree determination and population genetics. In closely related species such as bovids, heterologous polymerase chain reaction (PCR) primers may in some cases be used, bypassing the need to isolate and characterize microsatellite-containing sequences and design PCR primers. We report on the ability of a set of eighty bovine derived DNA microsatellite primers to amplify sequences in the two types (swamp and river) of water buffalo ( Bubalus bubalis ). Number of alleles and per cent heterozygosities in a large number of animals were determined on a subset of microsatellite loci selected on the robustness of the primers. These loci will form the basis of a set of polymorphic DNA markers for use in water buffalo.     

Endoreduplication of the hyperhaploid maternal complement and abnormal pronuclear formation in a human zygote obtained after intracytoplasmic sperm injection

We report the cytogenetic analysis of a tripronuclear zygote with two polar bodies observed after intracytoplasmic sperm injection. Rare previous investigations of this kind of zygote suggested a diploid or a hypotriploid chromosome constitution. In contrast, the present case turned out to be hypertriploid. Besides the haploid (23,Y) sperm chromosome set, there was a hyperdiploid endoreduplicated (end48,XX,+18,+18) maternal contribution. This zygote not only revealed a peculiar combination of different anomalies (hyperhaploidy of the female gamete, endoreduplication and abnormal pronuclear formation) but also indicates that endoreduplication may sporadically contribute to the generation of triploidy.     

Genetic differentiation of water buffalo (Bubalus bubalis) populations in China, Nepal and south-east Asia: inferences on the region of domestication of the swamp buffalo

Data from three published studies of genetic variation at 18 microsatellite loci in water buffalo populations in China (18 swamp type, two river type), Nepal (one wild, one domestic river, one hybrid) and south-east Asia (eight swamp, three river) were combined so as to gain a broader understanding of genetic relationships among the populations and their demographic history. Mean numbers of alleles and expected heterozygosities were significantly different among populations. Estimates of θ (a measure of population differentiation) were significant among the swamp populations for all loci and among the river populations for most loci. Differentiation among the Chinese swamp populations (which was due primarily to just one population) was much less than among the south-east Asian. The Nepal wild animals, phenotypically swamp type but genetically like river type, are significantly different from all the domestic river populations and presumably represent the ancestral Bubalus arnee (possibly with some river-type introgression). Relationships among the swamp populations (D(A) genetic distances, principal component analysis and structure analyses) show the south-east Asian populations separated into two groups by the Chinese populations. Given these relationships and the patterns of genetic variability, we postulate that the swamp buffalo was domesticated in the region of the far south of China, northern Thailand and Indochina. Following domestication, it spread south through peninsular Malaysia to Sumatra, Java and Sulawesi, and north through China, and then to Taiwan, the Philippines and Borneo.     

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection

Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2 g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F_2α was given on Days 6 and 7 after aspiration. A follicle ≥25 mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.     

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.     

Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig

The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 °C for up to 48 h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Cζ (PLCζ) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCζ in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18 h at 24 °C (78%, 62%, and 35%, respectively) and for 48 h at 14 °C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCζ in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.     

In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer

We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

Repeated oocyte pick up in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH superstimulation

The objective in this study was to investigate the effect of repeated oocyte collection by transvaginal, ultrasound-guidance, oocyte pick-up (OPU) in nine, prepubertal (8-12 months), swamp buffaloes. Animals were treated with FSH for 3 days and received GnRH on the third day, 24 h before OPU. This session was repeated on five occasions at 2 weekly intervals. Over the five sessions of hormone treatment followed by OPU, 39/42 (92.9%) animals responded and had 6.6+/-3.6 follicles with a follicular diameter of 5.0+/-2.0 mm. The oocyte recovery rate was 5.4+/-3.7 and averaged 82.8% oocytes, except for session 4, when oocyte recovery was around 75.0%. Most oocytes were denuded (39.5%), whilst 28.8% had a substantial cumulus mass. There were no differences in the ovarian responses and the recovery rates between the collections. It was concluded that five repeat cycles of FSH and OPU did not influence the follicular response to superstimulation or the number of oocytes recovered from prepubertal, buffalo calves.     

Developmental failure of hybrid embryos generated by in vitro fertilization of water buffalo (Bubalus bubalis) oocyte with bovine spermatozoa

The developmental potential of inter-species hybrid embryos produced by in vitro fertilization of in vitro matured buffalo oocytes with bovine spermatozoa was studied with a view to investigate pre-implantation embryo development and its gross morphology, early embryonic gene expression, and embryonic genome activation. Fertilization events with both buffalo and cattle spermatozoa were almost similar. Overall fertilization rate with cattle spermatozoa was 78.4% was not significantly different from that of buffalo spermatozoa (80.2%). Initial cleavage rate between buffalo and hybrid embryo was also similar, and there was no significant difference in their developmental rate till 8-cell stage (26.0 +/- 4.1 vs. 24.3 +/- 4.8). However, only 5.3% of hybrid embryos developed into blastocyst stage compared to 21.7% in buffalo. mRNA phenotyping of insulin-like growth factor family (Insulin, insulin receptor, IGF-I, IGF-I receptor, IGF-II, and IGF-II receptor) and glucose transporter isoforms (GLUT-I, II, III, IV) in hybrid embryos clearly showed that these molecules were not expressed after 8-cell stage onward. Similarly, as observed in buffalo embryos, incorporation of (35)S-methionine and (3)H-uridine could not be observed in hybrid embryos from 8-cell stage onward. This suggests that the maternal-zygotic genome activation did not occur in hybrid embryos. Differential staining also showed that the blastomere stopped dividing after 8-cell stage. Collectively, these parameters clearly showed that there was developmental failure of hybrid embryos.     

Glutathione and cysteine enhance porcine preimplantation embryo development in vitro after intracytoplasmic sperm injection

Because intracytoplasmic sperm injection (ICSI) had been introduced to animal science, not only reproductive biology of domestic animals, but also medicine to treat infertility has been developed. This assisted reproductive technology is beneficial for generating transgenic animals, especially pigs, because polyspermy is the greatest hurdle in porcine IVF when researchers make highly qualified preimplantation embryos. However, ICSI-derived embryos expressed high level of reactive oxygen species (ROS), which are known to cause serious dysfunction during preimplantation development. The objective of this study was to investigate the developmental competence, ROS level, and apoptosis index when glutathione (GSH) or cysteine was supplemented into the in vitro culture medium for ICSI-derived porcine embryos. First, we evaluated the effect of different concentrations of GSH or cysteine on developmental ability of porcine ICSI-derived embryos. The cleavage rate (79.6%) and the blastocyst formation rate (20.9%) were significantly improved in culture medium supplemented with 1 mmol/L GSH compared with other concentrations or no supplementation. Also, 1.71 mmol/L cysteine showed a significantly higher proportion of cleavage (80.7%) and blastocyst formation (22.5%) than other cysteine-supplemented groups. Next, we confirmed that intracellular ROS level was significantly reduced in the group of blastocysts cultured with GSH or cysteine after ICSI compared with the no supplementation group. Finally, we found that terminal uridine nick-end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased in blastocysts when ICSI-derived embryos were cultured with supplementation of 1.71 mmol/L cysteine or 1 mmol/L GSH. Taken together, these results strongly indicate that GSH or cysteine can improve the developmental competence of porcine ICSI-derived embryos by reducing intracellular ROS level and the apoptosis index.     

Isolation and characterization of EG-like cells from Chinese swamp buffalo (Bubalus bubalis)

There have been few studies done on the isolation and characterization of Chinese swamp buffalo embryonic germ cells (EG cells). Here, we first report on EG-like cells isolated from Chinese swamp buffalo fetuses. The results showed the cells grew in large, multilayered colonies, which were densely packed with an obvious border resembling mouse embryonic stem cells (ES cells) and EG cells. The buffalo EG-like cells expressed AP, SSEA-1, SSEA-3, SSEA-4 and OCT-4. By RT-PCR, we found that undifferentiated swamp buffalo EG-like cells expressed the OCT-4, NANOG, SOX2, FOXD3, GP130, STAT3, and HEB gene mRNA, but not Fgf4. When these cells were cultured for more than 2weeks without passage, they could differentiate into several types of cells including fibroblast-like, neuron-like, smooth muscle-like, and epithelial-like cells. Some cells formed simple embryoid bodies (EBs) and cystic EBs by suspension culture. By RT-PCR, we found cystic EBs expressed FOXD3, GP130, STAT3 and HEB gene mRNA, but not OCT-4, NANOG, and SOX2 gene mRNA, which could be detected in undifferentiated buffalo EG-like cells. At the same time, the expression of KERATIN-14 (Endoderm), GATA4, ACTA2 (Mesoderm) and TUBB3 (Ectoderm) gene mRNA were also detected in cystic EBs. The results suggested that these cells were capable of forming three germ layers in in vitro differentiation. The expression of OCT-4, NANOG and SOX2 might be essential for Chinese swamp buffalo EG-like cells in a pluripotent state. During the isolation and culture of Chinese swamp buffalo EG-like cells, we found the fetuses that were at 30-80days post-coitus were more efficient than others; and the mechanical method was better than trypsin digestion. The maximal passage of the mechanical method was eight, but the trypsin digestion was just three passages. So it seemed like that the buffalo EG-like cells were sensitive to trypsin. In summary, we were the first to isolate and characterize Chinese swamp buffalo EG-like cells that had morphology and characterization similar to those of established EG/EG-like cells in mouse and human.     

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 μM, Caffeic acid 100 μM, and Caffeic acid 200 μM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 μM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 μM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 μM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 μM Caffeic acid enhances the semen quality of water buffalo after thawing.     

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.     

Birth of twins after in vitro fertilization with flow-cytometric sorted buffalo (Bubalus bubalis) sperm

Flow-cytometric sorting of mammalian sperm for production of offspring with the desired sex is one of the most important new biotechnologies available for livestock industry. The objectives of this study were: (i) to sort the sperm into X- and Y-enriched populations and (ii) use the sorted sperm for in vitro fertilization (IVF) to produce sex-preselected embryo and offspring. The results revealed that the accuracy of sorted X- and Y-sperm was 94% and 89%, respectively. There was a decrease in blastocyst development rate in IVF with sorted sperm comparing to unsorted sperm, but the percentage of blastocysts on D6-D8 was not statistically different. Transplantation of the presumed X-embryos derived from IVF into a recipient resulted in the birth of female twins. These results indicated the feasibility of sperm sorting by flow cytometry and in vitro production of sex-preselected embryos and offspring in buffalo.