Assessment of Different Functional Parameters of Frozen–Thawed Buffalo Spermatozoa by Using Cytofluorimetric Determinations

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen–thawed buffalo spermatozoa quality parameters such as sperm viability by SYBR‐14/propidium iodide staining; mitochondrial function by JC‐1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC‐PNA staining. Semen samples from five Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Δψ^high), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five buffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %‐DFI, ‐DFI, SD‐DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage of acrosome‐reacted live (ARL) and acrosome‐reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca²⁺ ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functional parameters sensitive enough for the diagnosis of frozen‐thawed semen fertilizing potential.     

Capacitation of Frozen Thawed Buffalo Bull (Bubalus bubalis) Spermatozoa with Higher Heparin Concentrations

The present study was conducted to examine the effect of high heparin concentration on capacitation of buffalo spermatozoa with a short incubation time. Frozen thawed spermatozoa from three buffalo bulls were pooled and treated with either 50, 100 or 200 microg/ml heparin for 30 min. Capacitation was evaluated by acrosome reaction of spermatozoa and in vitro fertilization rate (per cent cleavage rate, per cent cleavage index). Acrosome reaction was induced in heparin treated spermatozoa with calcium ionophore A23187 and staining was carried out with Coomassie G-250 to evaluate the response as compared with control (0 heparin + calcium ionophore). Significantly higher percentage of acrosome reaction (AR) spermatozoa was noted after heparin treatment (36.8-48.2%) as compared with control (8.1% ; p < 0.05) but differences among the three heparin concentrations were non-significant. However, a significantly higher in vitro fertilization rate was recorded in spermatozoa capacitated by 50 and 100 microg/ml heparin (80.4 and 75.9% cleavage rate, respectively) as compared with 200 microg/ml heparin (47.2% cleavage rate; p < 0.001). It is concluded that buffalo spermatozoa capacitated with 50-100 microg/ml heparin had significantly higher ability to improve in vitro fertilization rate in buffalo.     

Ovarian Follicular Dynamics in Buffalo Cows (Bubalus bubalis)

Follicular growth in Egyptian buffalo cows was monitored using genital tracts from 200 buffalo cows collected immediately after slaughter. According to the morphological appearance of the corpus luteum (CL), the corresponding oestrous cycle was divided into four stages: A (days 1–4), B (days 5–10), C (days 11–17) and D (days 18–21). Within these stages the follicular population on the ovaries was evaluated and the dominant follicle (DF) determined in all recovered ovaries. The functional status of the DF and the largest sub‐dominant follicles was examined by histological examination in 31 cases, and Radio Immunoassay (RIA) analyses for estradiol‐17β (E₂) and progesterone (P₄) was performed in the follicular fluid in 23 of the DF. The results showed that DFs changed their endocrine character within the stages of the oestrous cycle. The DFs between days 5 and 10 were functionally active (E₂‐dominant; non‐atretic) in most of the cases. Between days 11 and day 17 half of the DFs became functionally inactive (P₄‐dominant; atretic). At days 18–21 all of the DF became functionally active and non‐atretic. In the specimens that carried two large follicles one of them was regularly atretic and P₄‐dominant whereas the other was non‐atretic and E₂‐dominant. Between days 18 and 21 all ovaries examined showed at least one large follicle. These findings suggest that in most of the cases follicular dynamics occurs in two wave‐like patterns in the Egyptian buffalo cows.     

Effect of Incorporation of Additives in Tris‐Based Egg Yolk Extender on Buffalo (Bubalus bubalis) Sperm Tyrosine Phosphorylation During Cryopreservation

Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4‐bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris‐based egg yolk extender supplemented with additives like taurine (50 mm ) or trehalose (100 mm ) or 4‐bromophenacyl bromide (200 μm ) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen‐thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris‐based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho‐proteins using SDS–PAGE followed by immunoblotting. Monoclonal anti‐phosphotyrosine antibody (Clone pT‐154) was used as primary antibody followed by treatment with HRP‐conjugated secondary antibody. Signals were detected on X‐ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post‐thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.     

Retracted: Influence of Glutamine Supplementation on Motility and Fertilization Success of Frozen–Thawed Persian Sturgeon (Acipenser persicus) Sperm

Amino acids have an important biological role for the prevention of cell damage during cryopreservation. The aim of this study was to investigate the effects of glutamine on post‐thaw sperm motility and fertilization success in the Persian sturgeon (Acipenser persicus). Sperm collected from six fish was cryopreserved in extenders containing different glutamine concentrations (2.5, 5 and 10 mm ). Sperm samples diluted at the ratio of 1 : 1 using the extenders were subjected to cryopreservation. After dilution, the sperm suspensions were sucked into 250‐μl straws; the straws were placed on the tray, frozen in nitrogen vapour and plunged into liquid nitrogen. Then, sperm were thawed in a water bath at 40°C for 5 s and used for analysis. Our results revealed that an increase in the concentration of glutamine caused a significant increase in the motility percentage, curvilinear velocity (VCL) and also fertilization success in the Persian sturgeon (p < 0.05). Comparing all concentrations of glutamine, the best concentration for sperm motility and fertilization rate was 10 mm . In addition, higher post‐thaw motility percentage, VCL, and fertilization and hatching rates were obtained with the extender at the concentration of 10 mm (p < 0.05). The findings of this study showed that glutamine was of greater benefit to Persian sturgeon sperm motility during frozen–thawed process.     

In Vitro Culture and Differentiation of Buffalo (Bubalus bubalis) Spermatogonia

The objective of this study was to develop a culture system which could support buffalo spermatogonia differentiation into spermatids in vitro. Testes from 3‐ to 5‐month‐old buffaloes were decapsulated and seminiferous tubules were enzymatically dissociated to recover spermatogonia and sertoli cells. The cells were cultured in modified Dulbecco modified Eagle medium supplemented with different concentrations of foetal bovine serum, retinol, testosterone for 2 months at 37°C. Spermatogonia and sertoli cells were identified with an antibody against c‐kit or GATA4, respectively. The viability of spermatogonia in the media supplemented with different concentrations of serum was all significantly higher (p < 0.05) compared with that in the medium without serum. A‐paired or A‐aligned spermatogonia and spermatogonial colonies (AP‐positive) were observed after 7–10 days of culture and spermatid‐like cells with a flagellum (6–8 μm) appeared after 30 days of culture. For cultured conditions, retinol could not significantly promote the formation of spermatid‐like cells (p > 0.05), whereas supplementation of testosterone could significantly promote (p < 0.05) the formation of spermatid‐like cells after 41 days of culture. The expression of the spermatid‐specific marker gene (PRM2) was identified after 30 days of culture by RT‐PCR. Yet, the transition protein 1 (TP1, a haploid makers) was not detected. Meanwhile, spermatids developed in vitro were also confirmed by Raman spectroscopy. These results suggest that buffalo spermatogonia could differentiate into spermatids in vitro based on the analysis of their morphology, PRM2 expression and Raman spectroscopy. Yet, the normality of the spermatid‐like cells was not supported by TP1 expression.     

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

Comparison of DNA Fragmentation Assay in Frozen‐Thawed Cat Epididymal Sperm

DNA fragmentation of frozen‐thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA^®), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis.     

Production of Wild Buffalo (Bubalus arnee) Embryos by Interspecies Somatic Cell Nuclear Transfer Using Domestic Buffalo (Bubalus bubalis) Oocytes

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin‐18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open‐pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re‐expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified–warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.     

Evidence of Lamina muscularis mucosae in Rumen of Buffalo (Bubalus bubalis)

Histological sections were studied from 4 sites of the rumen of 22 buffaloes, aged from 1 day to over 18 years of age. The sections were stained with Masson's trichrome stain. A definite layer of smooth muscle cells, representing the lamina muscularis mucosae separating the propria from the submucosa and extending into the ruminai papillae, was observed in buffaloes over 1.5 years of age. In animals over 10 years, the smooth muscle cells were very thin and elongated.     

Molecular Characterization of Oxytocin Receptor Gene in Water Buffalo (Bubalus bubalis)

Buffaloes are known for their productivity as compared to average yielding cows due to higher fat percentage, better feed conversion ability and disease resistance. On the other hand, the reproductive performances of buffaloes are often considered as poor owing to late sexual maturity, weak/silent oestrus, repeat breeder and prolonged intercalving interval. The study of cascade of events during oestrus and oestrous cycle can be useful for the improvement of reproductive efficiency of buffaloes. More precisely, the hormonal changes initiated at the molecular level within the animal determine the reproductive nature of the species. Nucleotide/protein sequence analysis serves as a vital tool in analysing the binding of the hormones for their effect or functions. In this study, we have reported cloning and characterization of the complete coding (cDNA) sequence of oxytocin receptor gene (OXTR) in buffaloes. Buffalo OXTR gene contains an uninterrupted ORF of 1176 nucleotides corresponding to an inferred polypeptide length of 391 amino acids (aa). The molecular weight of the deduced aa sequence was found to be 43 kDa with an isoelectric point of 9.253 and 16.328 charge at pH 7.0. The deduced protein sequence consists of 38 strongly basic (+) (K,R), 22 strongly acidic (?) (D,E), 186 hydrophobic (A, I, L, F, W, V) and 95 Polar (N, C, Q, S, T, Y) aa. Results indicated that aspartate (D) at aa position 85 and D, R and C at aa positions 136, 137 and 138, respectively, are conserved in buffaloes. The buffalo OXTR gene shared a per cent similarity ranging from 84.7 to 98.1 and 88.5 to 97.7 at nucleotide and deduced aa sequence levels, respectively, with that of other species. Phylogram constructed on the basis of either nucleotide or deduced aa sequences of buffalo OXTR gene showed that buffalo, cattle and sheep have diverged from human and swine and formed a separate clad. The buffalo sequence has shown maximum similarity and closeness with cattle followed by sheep both at nucleotide and at aa level.     

Factors Affecting the Quality of Cryopreserved Buffalo (Bubalus bubalis) Bull Spermatozoa

Storage of buffalo ( Bubalus bubalis ) bull semen in the cryopreserved state is discussed in this article. Fertility rate in buffalo following artificial insemination with frozen–thawed semen is reviewed. To better understand the freezability of bubaline spermatozoa, the available data on biochemical components and the activity of specific enzymes of semen/spermatozoa are given. Moreover, the major factors that may influence the post-thaw viability and fertility of buffalo spermatozoa are examined in detail. In addition, suggestions for improvement in cryogenic procedures for buffalo spermatozoa are also given.     

中国水牛遗传育种研究进展

我国水牛遗传育种的研究主要集中在染色体核型、蛋白质多态性、水牛胚胎体外生产与胚胎移植技术等方面。本文从中国水牛的类型和分布特征、遗传特性的研究现状、现代生物技术在水牛育种中的应用、中国水牛育种研究前景及展望等四个方面综述水牛的育种进展,认为以现代生物技术为核心的分子育种将成为水牛育种的总趋势,为我国水牛的产业化发展提供了理论依据。     

Production of Buffalo (Bubalus bubalis) Embryos in vitro: Premises and Promises

Techniques for in vitro production (IVP) of buffalo embryos adopting the procedures developed in cattle have received increasing interest in the recent times. A high oocyte maturation, fertilization and cleavage rate and a low rate of blastocyst yield and calving following transfer of in vitro produced buffalo embryos have been obtained. The efficiency of IVP in buffalo is much lower than that in cattle. Several problems need to be resolved before IVP technology can be used regularly in buffalo breeding. This review attempts to present an overview of the different techniques used in buffalo to produce transferable embryos in vitro, namely in vitro maturation and fertilization of immature oocytes and in vitro development of the resulting cleaved embryos to the blastocyst stage before transfer. The problems associated with IVP, the possible solutions and the new biotechniques linked to IVP are discussed.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

Effect of Antibiotics in Extender on Bacterial and Spermatozoal Quality of Cooled Buffalo (Bubalus bubalis) Bull Semen

The present study was designed to study the effect of traditional antibiotic combination (streptomycin and penicillin; SP) and relatively modern combination of antibiotics (gentamycin, tylosin, lincomycin and spectinomycin; GTLS) in extender on bacterial control and spermatozoal quality of liquid buffalo bull semen stored at 5°C. Semen collected from Nili‐Ravi buffalo bulls (n = 10) was diluted with skim milk extender containing either SP (streptomycin 1000 μg/ml and penicillin 1000 IU/ml), GTLS (gentamycin 500 μg/ml, tylosin 100 μg/ml, lincomycin 300 μg/ml and spectinomycin 600 μg/ml) or negative control with no antibiotics (NA). Liquid semen was stored at 5°C for 5 days. Aerobic bacteria isolated from buffalo semen were Pseudomonas aeruginosa and Staphylococcus aureus. The only facultative anaerobic bacterium isolated was Klebsiella pneumoniae. In vitro antibiotic sensitivity test revealed that Ps. aeruginosa and Staph. aureus were susceptible to gentamycin. Staphylococcus aureus and K. pneumoniae were susceptible to tylosin and linco‐spectinomycin. Total aerobic bacterial count was significantly lower in semen samples treated with GTLS than those of SP on third and fifth day of storage at 5°C. There was no difference (p > 0.05) in sperm motility, longevity and plasma membrane integrity (PMI) in extender containing SP or GTLS combination until the third day of storage at 5°C. On fifth day of storage sperm motility, longevity and PMI was significantly better in extender containing SP compared with GTLS and NA. Intact acrosomes, and sperm head, mid piece and tail abnormalities remained similar (p > 0.05) because of antibiotics up to 5 days of storage. In conclusion, GTLS is more capable than SP for bacterial control of buffalo bull semen. Moreover, GTLS and SP are equally efficient in preserving spermatozoal quality of extended buffalo bull semen for 3 days at 5°C.     

Effects of Chemical Activation Treatment on Development of Swamp Buffalo (Bubalus bubalis) Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Injection

The objective of this study was to optimize the activation protocol for buffalo oocytes after intracytoplasmic sperm injection (ICSI). The release of the second polar body (PB) at 3, 6 and 9 h after ICSI of in‐vitro matured oocytes activated either with 5 μm ionomycin (Io) or with 7% ethanol (EtOH) was preliminary examined. The highest rate of second PB extrusion occurred at 3 h of activation, and the second PB extrusion in EtOH group was significantly higher than that in Io group. Oocytes that extruded the second PB were selected and cultured either with 1.9 mm 6‐dimethylaminopurine (6‐DMAP) for 3 h or with 10 μg/ml cycloheximide (CHX) for 5 h. Significantly higher rate of oocytes formed 2 pronuclei in EtOH combined with CHX (EtOH + CHX) (62%) group compared to those of Io + CHX (42%) and EtOH + 6‐DMAP (48%) groups (p < 0.01) whereas Io + 6‐DMAP group showed intermediate value (58%). Significantly higher blastocyst formation rates were obtained in Io + 6‐DMAP (29%) and EtOH + CHX (24%) groups than in Io + CHX (6%) and EtOH + 6‐DMAP (17%) groups. Our results indicate that buffalo ICSI oocytes are effectively activated by combination treatment of Io with 6‐DMAP and EtOH with CHX resulting in the highest cleavage and blastocyst formation rates.