Sperm chromatin integrity is vital for successful pregnancy and transmission of genetic material to the offspring. We evaluated chromatin integrity in sperm from 60 infertile men and 7 fertile donors comparing the sperm chromatin structure assay (SCSA), TdT-mediated-dUTP nick end labeling (TUNEL), the sperm chromatin dispersion (SCD) test, and acridine orange staining technique (AOT). The TUNEL and SCD assays showed a strong relationship with the SCSA (r Ͼ .866; P Ͻ .001) for sperm DNA fragmentation, both in infertile men and donors of known fertility. AOT did not show any relationship with SCSA. The breakdown of the DNA fragmentation index (DFI) into 3 categories (Յ15%, Ͼ15%-Ͻ30%, and Ն30%) showed that the SCSA, TUNEL, and SCD test predict the same levels of DNA fragmentation. AOT consistently showed higher levels of DNA fragmentation for each DFI category. DNA fragmentation in sperm between infertile men and donor sperm was significantly different (P Ͻ .05) under SCSA (22.0 Ϯ 1.6 vs 11.8 Ϯ 1.4), TUNEL (19.5 Ϯ 1.3 vs 11.1 Ϯ 0.9) and SCD (20.4 Ϯ 1.3 vs 10.8 Ϯ 1.1), respectively. DNA fragmentation in sperm evaluated by AOT did not differ (P Ͼ .05) between infertile men (31.3 Ϯ 2.4) and donors (32.7 Ϯ 4.8). AOT showed extreme variations for sperm DNA fragmentation in semen from both infertile men and donors. The problems of indistinct colors, rapid fading, and the heterogeneous staining were also faced. In conclusion, SCSA, TUNEL, and SCD show similar predictive values for DNA fragmentation, and AOT shows variable and increased levels of DNA fragmentation, which makes it of questionable value in clinical practice.

Fertility of spermatozoa depends on maintenance of the mitochondrial transmembrane potential (⌬ m), which is generated by the electron-transport chain and regulated by an oxidation-reduction equilibrium of reactive oxygen intermediates, pyridine nucleotides, and glutathione (GSH). Here, we report that male mice lacking transaldolase (TAL) ؊/؊ are sterile because of defective forward motility. TAL ؊/؊ spermatozoa show loss of ⌬m and mitochondrial membrane integrity because of diminished NADPH, NADH, and GSH. Mitochondria constitute major Ca 2؉ stores; thus, diminished mitochondrial mass accounts for reduced Ca 2؉ fluxing, defective forward motility, and infertility. Reduced forward progression of TAL-deficient spermatozoa is associated with diminished mitochondrial reactive oxygen intermediate production and Ca 2؉ levels, intracellular acidosis, and compensatory down-regulation of carbonic anhydrase IV and overexpression of CD38 and ␥-glutamyl transferase. Microarray analyses of gene expression in the testis, caput, and cauda epididymidis of TAL ؉/؉ , TAL ؉/؊ , and TAL ؊/؊ littermates confirmed a dominant impact of TAL deficiency on late stages of sperm-cell development, affecting the electrontransport chain and GSH metabolism. Stimulation of de novo GSH synthesis by oral N-acetyl-cysteine normalized the low fertility rate of TAL ؉/؊ males without affecting the sterility of TAL ؊/؊ males. Whereas TAL ؊/؊ sperm failed to fertilize TAL ؉/؉ oocytes in vitro, sterility of TAL ؊/؊ sperm was circumvented by intracytoplasmic sperm injection, indicating that TAL deficiency influenced the structure and function of mitochondria without compromising the nucleus and DNA integrity. Collectively, these data reveal an essential role of TAL in sperm-cell mitochondrial function and, thus, male fertility. F orward motility and fertility of spermatozoa depend on production of reactive oxygen intermediates (ROIs) (1) and maintenance of the mitochondrial transmembrane potential (⌬ m ) (2, 3). ⌬ m is generated by the electron-transport chain and subject to regulation by an oxidation-reduction equilibrium of ROI, pyridine nucleotides (NADH͞NAD ϩ NADPH͞NADP), and reduced glutathione (GSH) (4). In turn, NADPH, a reducing equivalent required for biosynthetic reactions and regeneration of GSH from its oxidized form, is produced by the pentose phosphate pathway (PPP) (5). The PPP was originally formulated based on metabolites and enzymes detected in yeast (6). Thus, PPP comprises two separate oxidative and nonoxidative phases. Enzymes of the oxidative phase, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase, can generate both ribose 5-phosphate (R5P) and NADPH. Although enzymes of the nonoxidative phase, transketolase (TK) and transaldolase (TAL), can convert R5P into glucose 6-phosphate (G6P) for the oxidative phase, and, thus, indirectly, these enzymes can also contribute to the generation of NADPH, the significance of the nonoxidative branch is less well established. Certain organisms (7,8) and mammalian tissu...

The buffalo is an important livestock resource in several countries of South Asia and the Mediterranean regions. However, reproductive efficiency is compromised due to known problems of biological and management origins, such as lack of animal selection and poor nutrition. Under optimal conditions puberty is attained at 15 to 18 months in river buffalo, 21 to 24 months in swamp buffalo and is influenced by genotype, nutrition, management and climate. However, under field conditions these values deteriorate up to a significant extant. To improve reproductive efficiency, several protocols of oestrus and ovulation synchronization have been adopted from their use in commercial cattle production. These protocols yield encouraging pregnancy rates of (30% to 50%), which are comparable to those achieved in buffaloes bred at natural oestrus. The use of sexed semen in buffalo heifers also showed promising pregnancy rates (50%) when compared with conventional non-sexed semen. Assisted reproductive technologies have been transferred and adapted to buffalo but the efficiency of these technologies are low. However, these latest technologies offer the opportunity to accelerate the genetic gain in the buffalo industry after improving the technology and reducing its cost. Most buffaloes are kept under the small holder farming system in developing countries. Hence, future research should focus on simple, adoptable and impact- oriented approaches which identify the factors determining low fertility and oestrus behaviour in this species. Furthermore, role of kisspeptin needs to be explored in buffalo.

Staining of cells with acridine orange (AO) has been widely accepted as a predictor of DNA damage in many cell types. Because of variability of protocols used in previous studies, the AO staining technique has not been widely accepted as a screening test to predict DNA damage in human sperm. In order to further validate the use of AO staining, sperm were evaluated using numerous variations in the staining protocol. This study also elucidated the effects of cryopreservation on sperm DNA. Sperm fixation in Carnoy's solution showed significantly (P < 0.05) more DNA damage (29.9 +/- 4.5%) than 2% glutaraldehyde (14.4 +/- 2.1%), 4% paraformaldehyde (5.5 +/- 1.7%), no fixation (15.8 +/- 4.3%) but did not differ from Diff Quik solution (19.2 +/- 5.8%). No difference was observed for sperm DNA damage assessment using a 0.2 m (15.5 +/- 3.2%) or 0.3 m (14.9 +/- 3.3%) concentration of Na(2)HPO(4).7H(2)O in the AO staining solution. Frozen-thawed semen samples showed increased damage to sperm DNA under both Carnoy's (fresh: 10.9 +/- 1.3%; frozen: 30.8 +/- 2.9%; P < 0.05) and Diff Quik fixation (fresh: 6.2 +/- 0.8; frozen: 17.1 +/- 2.5%P < 0.05). Present data also showed that spermatozoa from some individuals are more prone to DNA damage after freezing and thawing procedures than others. In conclusion, Carnoy's fixative provides a better predictive value for DNA damage to sperm using AO staining. Additionally, cryopreservation increased damage to the sperm DNA.

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The nuclear morphology, diameter and in vitro meiotic competence of buffalo oocytes was compared relative to follicle size. Cumulus-oocyte complexes (COCs) were collected from 1-<2, 2-<3, 3-<4, 4-<6 and 6-<8 mm follicles from abattoir ovaries. Cumulus cells were removed using 3 mg mL(-1) hyaluronidase in saline and repeated pipetting. Denuded oocytes were measured, fixed in 3% glutaraldehyde, stained with 4,6-diamidoino-2-phenylindole and evaluated for nuclear morphology, namely the stage of germinal vesicle (GV) development before in vitro maturation (IVM). The COCs from >2-mm follicles were matured in vitro in their respective size groups for 24 h in Medium 199 supplemented with 10 microg mL(-1) follicle-stimulating hormone, 10 microg mL(-1) luteinizing hormone, 1.5 microg mL(-1) oestradiol, 75 microg mL(-1) streptomycin, 100 IU mL(-1) penicillin, 10 mM HEPES and 10% fetal bovine serum. Matured oocytes were fixed, stained and evaluated for GV status and meiotic development. The number of oocytes collected from follicles 1-<8 mm in diameter averaged 1.82 per ovary. Oocytes from follicles 1-<2 mm (107.7 +/- 1.6 microm), 2-<3 mm (108 +/- 1.1 microm) and 3-<4 mm (114.6 +/- 1.3 microm) in diameter were smaller in diameter (P < 0.05) than oocytes from follicles 4-<6 mm (124.4 +/- 1.3 microm) and 6-<8 mm (131.9 +/- 1.4 microm) in diameter. A majority of oocytes (P< 0.05) from <4-mm follicles was at the initial stages of GV development (GV-I, II and III), whereas oocytes from 4-<6- and 6-<8-mm follicles were at the final stages of GV-IV (35.0 and 21.6% respectively) and GV-V (49.1 and 67.5% respectively). Poor IVM rates of 32.0% and 32.7% to metaphase (M)-II were observed for oocytes isolated from 2-<3- and 3-<4-mm follicles, respectively, whereas significantly (P< 0.05) more oocytes from 4-<6- and 6-<8-mm follicles reached M-II (67.1% and 79.1% respectively). In conclusion, buffalo oocytes displayed a size-dependent ability to undergo meiotic maturation and we suggest that oocytes from >4-mm follicles should be considered in buffalo in vitro fertilization systems for better results.