Muhammad Anzar

Cryopreservation of immature porcine testis tissue to maintain its developmental potential after xenografting into recipient mice.

The purpose of this study was to develop effective strategies for cooling and cryopreservation of immature porcine testis tissue that maintain its developmental potential. Testes from 1-wk-old piglets (Sus domestica) were subjected to 1 of 12 cooling/cryopreservation protocols: as intact testes, cooling at 4 degrees C for 24, 48, or 72h (Experiment 1); as fragments, programmed slow-freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol (Experiment 2); or solid-surface vitrification using DMSO, glycerol, or ethylene glycol, each using 5-, 15-, or 30-min cryoprotectant exposure times (Experiment 3). For testis tissue xenografting, four immunodeficient recipient mice were assigned to each protocol, and each mouse received eight grafts. Recipient mice were killed 16 wk after grafting to assess the status of graft development. Based on morphology and in vitro assessment of cell viability, cooling of testis tissue for up to 72h maintained structural integrity, cell viability, in vivo growth, and developmental potential up to complete spermatogenesis comparable with that of fresh tissue (control). In frozen-thawed testis tissues, higher numbers of viable cells were present after programmed slow-freezing using glycerol compared with that after DMSO or ethylene glycol (P<0.001). Among the vitrified groups, exposure to DMSO for 5min yielded numerically higher viable cell numbers than that of other groups. Cryopreserved tissue fragments recovered after xenografting had normal spermatogenesis; germ cells advanced to round and elongated spermatids after programmed slow-freezing using glycerol, as well as after vitrification using glycerol with 5- or 15-min exposures, or using DMSO for a 5-min exposure.

Effect of buffering systems on post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa

This study was carried out to identify the suitable buffer for cryopreservation of buffalo semen. Semen was collected with artificial vagina (42 degrees C) from four buffalo bulls. Split pooled ejaculates (n=5), possessing more than 60% visual sperm motility, were extended at 37 degrees C either in tri-sodium citrate (CITRATE), Tris-citric acid (TCA), Tris-Tes (TEST) or Tris-Hepes (HEPEST). Semen was cooled to 4 degrees C in 2 h, equilibrated at 4 degrees C for 4 h, filled in 0.5 ml straws and frozen in a programmable cell freezer before plunging into liquid nitrogen. Thawing of frozen semen was performed after 24 h at 37 degrees C for 15 s. Sperm motion characteristics, plasma membrane integrity, and acrosome morphology of each semen sample were assessed by using computer-assisted semen analyzer (CASA), hypo-osmotic swelling (HOS) assay, and phase-contrast microscope, respectively. Analysis of variance revealed that percent post-thaw visual motility tended (P=0.07) to be higher in HEPEST (61.0+/-2.9) and lowest in CITRATE (48.0+/-2.5). Computerized motility did not vary due to buffering system. Percent post-thaw linear motility tended (P=0.09) to be higher in TCA (78.2+/-5.5) and lower in TEST (52.0+/-6.9). Circular motility (%) was significantly lower (P<0.05) in TCA (11.6+/-2.8) and higher in TEST (29.8+/-5.6). Curvilinear velocity (microm s(-1)) was lower (P<0.05) in TCA (69.4+/-2.0) than in CITRATE (79.0+/-5.8), TEST (87. 2+/-1.6) and HEPEST (82.6+/-3.0). Lateral head displacement (microm) was lowest (P<0.05) in TCA (1.7+/-0.2) and highest in TEST (3.7+/-0. 6). Plasma membrane integrity and normal acrosomes of buffalo spermatozoa did not differ due to buffering system and averaged 40. 0+/-2.7% and 61.4+/-4.6%, respectively. Based upon lower circular motility, curvilinear velocity, and lateral head displacement, it is concluded that post-thaw quality of buffalo semen can be improved using the Tris-TCA buffering system.

Sephadex and sephadex ion-exchange filtration improves the quality and freezability of low-grade buffalo semen ejaculates

The effect of sephadex and sephadex ion-exchange filtration on the improvement in quality and freezability of low-grade buffalo semen ejaculates was assessed. Two types of filtration columns were used: one containing only sephadex G-10 (FS) and the other sephadex G-10 along with ion-exchangers (diethyl amino ethane-52 (DEAE-52) cellulose and carboxy methyl-52 (CM-52) cellulose; FS+IE). Unfiltered samples served as controls. Semen ejaculates extended in Tris-citric acid (1:4) (n=16; initial motility 40-50%) were filtered at the rate of 1.5 ml/min under negative pressure at room temperature (28-30 degrees C). The mean recovery rate (%) of motile spermatozoa in the FS (85.9+/-1.51) and FS+IE (77.10+/-2.28) filtrates did not differ significantly. Percentages of sperm motility, normal acrosomes, and intact plasma membranes were highest (P<0.05) in FS+IE, intermediate (P<0.05) in FS and lowest (P<0.05) in controls at the three stages of cryopreservation (postfiltration final dilution, after equilibration, and after freezing). Mean sperm abnormalities were lowest (P<0.05) in the filtrates of FS+IE, moderate (P<0.05) in FS and highest in controls at all stages of freezing. Compared to dilution and equilibration, freezing greatly reduced (P<0.05) the overall percent motility, normal acrosomes and intact plasma membranes. The spermatozoa eluted through FS+IE columns proved more resistant (P<0.05) in bearing dilution, equilibration, freezing and thawing stresses than the spermatozoa from FS and control samples. It is concluded that filtration systems containing an FS+IE column can effectively enhance the quality and freezability of extended, low quality buffalo semen.

Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.

Response of buffalo spermatozoa to low temperatures during cryopreservation

This is the first detailed report on the response of buffalo spermatozoa to low temperatures during freezing. The study determined the critical temperature zone for buffalo spermatozoa and developed a suitable freezing rate for this species. Semen from four Nili-Ravi buffalo bulls diluted in Tris-citric acid was frozen in a programmable freezer. Motion characteristics, plasma membrane integrity and acrosome morphology were determined at +4, 0, -5, -10, -20, -30, -40, -50, -80 and -196 degrees C by removing semen straws from the freezer at exactly these temperatures and rewarming them at 37 degrees C. The first statistical decline in sperm motility and lateral head displacement was observed at -40 degrees C. For all other parameters, there was biphasic decline: for curvilinear velocity, at 0 degrees C and -50 degrees C; and for plasma membrane integrity and acrosome morphology, at -30 degrees C and -50 degrees C. In a second series of experiments, buffalo spermatozoa were frozen using slow (-10 degrees C min(-1)), medium (-20 degrees C min(-1)) or fast (-30 degrees C min(-1)) freezing rates, between -10 degrees C and -80 degrees C. Freezing of buffalo spermatozoa at a rate of -30 degrees C min(-1) yielded higher post-thaw motion characteristics, plasma membrane integrity and normal acrosomes. In conclusion, different sperm characteristics respond differently at low temperatures and the freezing of buffalo spermatozoa at a higher rate ensures higher post-thaw semen quality.

Effect of in utero and lactational nicotine exposure on the male reproductive tract in peripubertal and adult rats

The objective of this study was to determine the effect of in utero and lactational exposure to nicotine on the male reproductive tract. Dams were randomly assigned to receive saline or nicotine bitartrate (1mg/kg-d s.c.) daily for two weeks prior to mating until weaning (postnatal day 21). Male offspring were sacrificed at 7 (peri-pubertal) and 26 (adult) weeks of age. Nicotine-exposure resulted in retention of spermatids after stage VIII, tubular vacuolation, degeneration of pachytene and round spermatids at stage VII in the testes; and lymphocyte infiltration, germ cell exfoliation, and hypospermia in epididymides, at 7 weeks of age. Nicotine-exposure had no effect on testis or epididymal morphology, daily sperm production, epididymal sperm reserve, sperm viability at 26 weeks of age, and circulating testosterone levels at either age examined. We conclude that maternal nicotine-exposure during pregnancy and lactation can induce transient structural changes in the testis and epididymis of male offspring.

Fertility-associated metabolites in bull seminal plasma and blood serum: 1H nuclear magnetic resonance analysis.

Early estimation of bull fertility is highly desirable for the conservation of male genetics of endangered species and for the exploitation of genetically superior sires in artificial insemination programs. The present work was conducted as a proof‐of‐principle study to identify fertility‐associated metabolites in dairy bull seminal plasma and blood serum using proton nuclear magnetic resonance (1H NMR). Semen and blood samples were collected from high‐ and low‐fertility breeding bulls (n = 5 each), stationed at Semex, Guelph, Canada. NMR spectra of serum and seminal plasma were recorded at a resonance frequency of 500.13 MHz on a Bruker Avance‐500 spectrometer equipped with an inverse triple resonance probe (TXI, 5 mm). Spectra were phased manually, baseline corrected, and calibrated against 3‐(trimethylsilyl) propionic‐2,2,3,3‐d4 acid at 0.0 parts per million (ppm). Spectra were converted to an appropriate format for analysis using Prometab software running within MATLAB. Principal component analysis was used to examine intrinsic variation in the NMR data set, and to identify trends and to exclude outliers. Partial least square‐discriminant analysis was performed to identify the significant features between fertility groups. The fertility‐associated metabolites with variable importance in projections (VIP) scores >2 were citrate (2.50 ppm), tryptamine/taurine (3.34–3.38 ppm), isoleucine (0.74 ppm), and leucine (0.78 ppm) in the seminal plasma; and isoleucine (1.14 ppm), asparagine (2.90–2.94 ppm), glycogen (3.98 ppm), and citrulline (1.54 ppm) in the serum. These metabolites showed identifiable peaks, and thus can be used as biomarkers of fertility in breeding bulls. Mol. Reprod. Dev. 82: 123–131, 2015.

Sperm survival kinetics in different types of bull semen: progressive motility, plasma membrane integrity, acrosomal status and reactive oxygen species generation

This study was designed to compare the kinetics of sperm survival in different types of bull semen. Fresh ejaculates from four bulls were pooled, diluted in Tris-citric acid-egg yolk-glycerol extender, cooled to 4°C, frozen in LN2 and thawed at 37°C. Fresh, diluted, cooled and frozen-thawed semen were incubated at 37°C, and evaluated at 0, 2, 4, 6, 12 and 24h after the beginning of incubation. In Experiment 1, progressive sperm motility, normal acrosomes and plasma membrane integrity and asymmetry were determined. In Experiment 2, generation of superoxide anion (O2(•)) along with plasma membrane permeability and generation of hydrogen peroxide (H2O2) along with plasma membrane integrity were assessed. In Experiment 1, frozen-thawed semen had shorter survival times for progressive sperm motility, and spermatozoa with intact plasma membranes and acrosomes (IPM-IACR) as compared with other types of semen (P<0.05). Fresh spermatozoa underwent a necrotic pathway, diluted and cooled spermatozoa underwent an apoptosis-like pathway and frozen-thawed spermatozoa underwent both necrotic and apoptosis-like pathways. In Experiment 2, spermatozoa in all four types of semen exhibited O2(•-) generation and increased plasma membrane permeability, and became necrotic without H2O2 generation during incubation (P<0.05). In conclusion, frozen-thawed semen had shorter sperm longevity, which has important implications relating to the timing of artificial insemination. Different types of semen followed different death pathways. During incubation, spermatozoa in all types of semen generated O2(•-), which increased the permeability and compromised the integrity of the plasma membrane.

Changes in the behaviour and androgen levels during pubertal development of the buffalo bull

Sexual behaviour and peripheral testosterone concentrations were studied longitudinally in a group of ten buffalo bulls of Nili-Ravi breed raised from birth under similar feeding and management conditions. Changes in sexual behaviour of these animals in response to a teaser bull were observed at weekly intervals from 15 months of age to attainment of puberty (mean age at puberty, 23.6 ± 0.9 months). Sexual behaviour of individual bulls was scored by a procedure based on an assessment of behavioural events and expressed as a percentage of the maximal points. These events ranged from reaction of the bull during approach towards the teaser to intromission and ejaculation in an artificial vagina. Serum testosterone was determined by radioimmunoassay in blood samples obtained at monthly intervals, from each bull. Analysis of behavioural data indicated a significant increase in sexual behaviour score (from 5 ± 2% to 40 ± 7%) 4–5 months prior to the attainment of puberty. Following a transitory decline, the sexual behaviour score reached a maximum (69 ± 5%) during the month in which first ejaculation with motile sperm was achieved. Behavioural profiles of individual bulls indicate that the period of prepubertal increase in sexual activity corresponded to the physiological puberty rather than to the chronological age of the bulls. The prepubertal as well as pubertal increase in sexual behaviour score was invariably preceded by a significant (P < 0.05) elevation of serum testosterone concentrations. The data suggests that the prepubertal increase in sexual activity in the buffalo bull may serve as a criterion of onset of puberty and may be helpful in the selection of sires at a stage prior to sexual maturation.

Relationship between sperm apoptosis and bull fertility: in vivo and in vitro studies

The objectives of this study were to confirm the relationship of apoptosis-associated membrane and nuclear changes in bull spermatozoa with field fertility, to predict the fertility of beef bulls used for natural breeding and to study the role of DNA-nicked spermatozoa in early embryonic development. In Experiment 1, the relationship between fertility and different sperm populations identified by the Annexin V/propidium iodide (PI) and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assays was determined. Bull fertility was related to live (PPPin vitro cleavage and blastocyst rates was evaluated, using 30000 or 300000 spermatozoa per droplet. Cleavage rate was adversely affected (PP<0.05) in high DNA-nicked spermatozoa at the lower sperm concentration. In conclusion, the incidence of DNA-nicked spermatozoa is a useful marker to predict a bulls fertility potential. DNA-nicked spermatozoa showed adverse effects on early embryonic development.