No abstract

The goal of this study was to define conditions for the successful isolation of embryonic stem cells from bovine blastocysts. Expression of the Pit-Oct-Unc (POU) transcription factor Oct4 was employed to monitor the pluripotent status of cultured cells. No expression of the previously identified bovine Oct4 pseudogene was found, and transcription of the Oct4 ortholog correlated with the proliferative potential of bovine ICM derived cells. Two methods to isolate pluripotent inner cell mass were compared; 90% of trypsin isolated ICMs formed growing cultures, whereas only 12%-23% of the ICMs isolated by immunosurgery attached and grew. Colony formation from complete blastocysts was 55%. The bovine ICM derived cells could be grown for 4-7 passages. However, Oct4 transcripts were only present in the primary cultures, indicating that the initial culture period of bovine ICM derived cells is critical and needs to be optimized to yield true ES cells. In contrast to bovine ICMs, murine ICMs yielded rapidly growing cells, which proliferated for more than 60 passages.

The present study was designed to investigate the sperm damages occurring in acrosome, plasma membrane, mitochondrial activity, and DNA of fresh, equilibrated and frozen-thawed buffalo semen by fluorescent probes. The stability of sperm acrosome and plasma membrane stability, mitochondrial activity and DNA status were assessed by fluorescein conjugated lectin Pisum sativum agglutinin, Annexin-V/propidium iodide, JC-1 and TUNEL assay, respectively, under the fluorescent microscope. The damages percentage of acrosome integrity was significantly increased during equilibration and freezing-thawing process. The stability of sperm plasma membrane is dependent on stability of phosphatidylserine (PS) on the inner leaflet of plasma membrane. The frozen-thawed sperm showed externalization of PS leading to significant increase in apoptotic, early necrotic and necrotic changes and lowered high mitochondrial membrane potential as compared with the fresh sperm but all these parameters were not affected during equilibration. However, the DNA integrity was not affected during equilibration and freezing-thawing procedure. In conclusion, the present study revealed that plasma membrane and mitochondria of buffalo sperm are more susceptible to damage during cryopreservation. Furthermore, the use of fluorescent probes to evaluate integrity of plasma and acrosome membranes, as well as mitochondrial membrane potential and DNA status increased the accuracy of semen analyses.

The seminal plasma is a mixture of secretions from the testis, epididymis and male accessory sex glands. A number of seminal plasma proteins are associated with male fertility but most of these have not been studied in detail till now. Recently, proteomics has been used to show the differences in proteins profile in seminal plasma from high and low fertility bulls. For example, osteopontin, phospholipase A2, P25b, acidic seminal fluid proteins, a-L-fucosidase and cathepsin D are positively correlated with fertility of bulls and may act as useful fertility marker (s), while lipocalin-type prostaglandin D synthase, spermadhesin Z13, clusterin and ubiquitin are negatively correlated with fertility in bulls. Bovine seminal plasma proteins in seminal plasma act like a double-edged sword and showed a quadratic association with bull fertility. The physiological roles of the metalloproteinase-2 (TIMP-2), ecto-ADP-ribosyltransferase 5, nuclobindin, Niemann-Pick C2 and epididymal sperm-binding protein 1 and their relationship to bull fertility need further studies. This review summarizes the physiological functions of proteins of seminal plasma and their relation to bull fertility.

Recent findings suggest that mammalian amniotic fluid (AF) is a source of multipotent stem cells (SCs), which can be used in regenerative medicine and assisted reproduction. We report the isolation, culture and characterization of amniotic fluid-derived cells from pregnant water buffalo uterus. These undifferentiated AF cells expanded without feeder layer over a period of 100 days up to passages 20 and the expression of alkaline phosphatase (AP), Oct-4, Nanog and Sox-2, GAPDH and β-actin could be detected by RT-PCR. The cells exhibited uniform morphology and normal chromosome number. The up-regulation or down-regulation of transcription factors of each gene varied with passage number. We conclude that putative bubaline AF cells can be cultured and maintained in vitro for a prolonged period and offer a potential source of multipotent cells for applications including assisted reproduction in buffalo.

Somatic cell nuclear transfer (SCNT) technology provides an opportunity to multiply superior animals that could speed up dissemination of favorable genes into the population. In the present study, we attempted to reproduce a superior breeding bull of Murrah buffalo, the best dairy breed of buffalo, using donor cells that were established from tail-skin biopsy and seminal plasma. We studied several parameters such as cell cycle stages, histone modifications (H3K9ac and H3K27me3) and expression of developmental genes in donor cells to determine their SCNT reprogramming potentials. We successfully produced the cloned bull from an embryo that was produced from the skin-derived cell. Growth, blood hematology, plasma biochemistries, and reproductive organs of the produced cloned bull were found normal. Subsequently, the bull was employed for semen production. Semen parameters such as CASA (Computer Assisted Semen Analysis) variables and in vitro fertilizing ability of sperms of the cloned bull were found similar to non-cloned bulls, including the donor bull. At present, we have 12 live healthy progenies that were produced using artificial insemination of frozen semen of the cloned bull, which indicate that the cloned bull is fertile and can be utilized in the buffalo breeding schemes. Taken together, we demonstrate that SCNT can be used to reproduce superior buffalo bulls.

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high-fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high-fertile bulls was found to be higher (p < .05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p < .05) in high-fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r = .678, p = 0.013) and TAC (r = .648, p = .042) were found to be positively correlated with bull fertility and the level of MDA (r = -.718, p = .019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.

Stem cells present an important tool in livestock assisted reproduction and veterinary therapeutic field such as tissue engineering. We report for the first time isolation of pluripotent stem cell-like cells expressing pluripotency markers (alkaline phospahatase, OCT-4, NANOG and SOX-2) from the amnion of water buffalo (Bubalus bubalis). The cells showed no apparent abnormalities in their chromosomal profiles before and after cryopreservation. The cytochemical staining revealed that pluripotent cells were capable of undergoing directed differentiation in vitro into osteocytes. It could be inferred that amnionderived pluripotent stem cell-like cells can be isolated, cultured for many passages and differentiated into mesoderm lineage, and may be an alternative source to mesenchymal stem cells. These cells can have applications in assisted reproduction, developmental biological and regenerative medicine.