In vitro development of bovine embryos cultured with stem cell factor or insulin-like growth factor-I following IVF with semen of two bulls having different field fertility

The usefulness of IVF as a potential tool to evaluate the field fertility of bulls is equivocal and growth factor addition to culture media research is needed to delineate components needed for providing defined environments for embryos. The overall aim was to evaluate the in vitro development of embryos derived using a serum supplemented and serum-free production systems and semen from two bulls of different field fertility. The study was conducted to determine the combinatorial effect of stem cell factor (SCF) and/or insulin-like growth factor-I (IGF-I) in culture on subsequent embryo development in cattle. Oocytes were aspirated separately from ≥3 to <3 mm follicles to test different follicle size populations and were matured in TCM-199 supplemented with LH, FSH, estradiol and BSA (Fraction V). Matured oocytes were fertilized in BSA supplemented synthetic oviductal fluid (SOF)-IVF medium. Presumptive zygotes were cultured for 8 d (in humidified 5% CO₂ at 38.5 °C) in BSA supplemented SOF-in vitro culture (IVC) medium. SOF-IVC medium was supplemented with fetal bovine serum (4%), IGF-I (100 ng/mL), SCF (50 ng/mL) or IGF-I (100 ng/mL) + SCF (50 ng/mL). The development competence of embryos did not differ between the bulls and among the culture environments. Nevertheless, there was an effect of follicle size on cleavage rate (P < 0.05) and a greater cleavage rate resulted from oocytes aspirated from ≥3 mm follicles (71.0 ± 1.5%) compared to those collected from <3 mm follicles (64.8 ± 1.6%). The overall cleavage rate (%); blastocyst formation (%); and expanded/hatched blastocyst formation (%) were 68.2 ± 1.5 and 67.7 ± 1.7; 29.4 ± 1.4 and 28.6 ± 1.5; and 18.6 ± 1.2 and 18.5 ± 1.1, respectively, for the bull of above and below average field fertility. The results indicate that follicle size for oocyte aspiration is effective for determining IVC success and that IVF may not discriminate among bulls of different field fertility.     

Collection and characterization of semen in Mithun (Bos frontalis) bulls

The objective of this study was to collect semen from semiwild Mithun (Bos frontalis) bulls using an artificial vagina (AV) and to determine semen characteristics. Collection of semen with an AV was attempted in five Mithun bulls using both anestrous and estrous Mithun females. No Mithun bull mounted an anestrous female Mithun during 60 trials, but satisfactory mounting, including extension of the penis, occurred in 25 trials with estrous Mithun females. In 15 of these trials, semen was successfully collected in an AV with an internal temperature of 42 to 46 °C. However, in 10 trials with an AV with an internal temperature of 36 to 40 °C, semen was not collected. Mean (± SEM) intervals to first mount and to ejaculation in the AV were 27.9 ± 3.6 sec and 113.8 ± 6.6 sec, respectively. Semen volume and pH were 3.1 ± 0.35 mL and 6.59 ± 0.04, and mean mass activity (scale, 0 to 4), initial sperm motility, live sperm count, sperm concentration, total number of sperm in the ejaculate, and overall sperm length were 2.2 ± 0.3, 78.6 ± 2.6%, 80.7 ± 2.2%, 710.8 ± 66.8 × 10⁶/mL, 2114 ± 364.4 sperm, and 67.9 ± 0.6 μm, respectively. The proportion of morphologically normal sperm was 80.6 ± 0.2%, whereas the proportion with a morphologically abnormal head, midpiece, tail, and acrosome were 4.2 ± 0.4%, 1.6 ± 0.5%, 6.1 ± 1.1%, and 7.1 ± 0.9%, respectively. The mean incidence of tail-less heads and proximal and distal protoplasmic droplets were 0.5 ± 0.1%, 0.3 ± 0.2%, and 2.4 ± 0.3%, respectively. In conclusion, we successfully collected semen from semiwild Mithun bulls with an AV maintained at 42 to 46 °C, and overall, the semen was within the normal range of that collected from fertile domestic bulls.     

Relationship between embryo development in vitro and 56-day nonreturn rates of cows inseminated with frozen-thawed semen from dairy bulls

Frozen-thawed bull semen with > 50% post-thaw motility from 40 batches (21 bulls, 2 consecutive ejaculates per batch) was used for fertilization (IVF) and embryo development in vitro to assess the relationship between field and laboratory fertility using a retrospective approach. Each frozen batch was tested in 3 or 4 replicates with 30 oocytes per replicate. Field fertility, quantified as the 56-d nonreturn rate and based on 89 to 441 artificial inseminations per frozen batch, ranged between 46.2 and 74.8%. The cleavage and blastocyst rates after IVF varied from 29.0 to 81.9% and from 1.8 to 32.0%, respectively, with significant differences among frozen batches. Rates of cleavage and blastocyst formation were significantly related to the nonreturn rate (r = 0.59, P < 0.001; r = 0.35, P < 0.05, respectively). The interaction between cleavage and blastocyst rate was 0.69 (P < 0.001). Significant variations (P < 0.05) among frozen semen batches within 15 bulls with >/= 2 different semen batches were found for the nonreturn rate (13.3%) of 2 bulls, for cleavage rates (26.7%) in 4 bulls and for blastocyst rates (20.0%) in 3 bulls. Significant differences (P < 0.05) among replicates within the 40 frozen semen batches were only found in 3 batches (7.5%) for the cleavage rate and in 7 batches (17.5%) for blastocyst rate. Overall, bull and frozen semen batch were the greatest sources of variation in the cleavage rate (30.6 and 29.4%, respectively), while testing date was the greatest source of variation in the blastocyst development rate (21.7%). The results indicated that in vitro fertilization and, to a lesser extent, culture to the blastocyst stage could be useful in estimating the potential fertilizing ability of frozen-thawed semen from dairy bulls.     

Spirulina platensis extract addition to semen extender enhances cryotolerance and fertilizing potentials of buffalo bull spermatozoa

The objective of this study was to investigate the effect of Spirulina platensis extract (SPE) addition to the freezing extender on freezability, lipid peroxidation, ultrastructure alterations and fertilizing potentials of frozen-thawed buffalo bull spermatozoa. Semen samples were collected with artificial vagina from five adult fertile bulls and diluted with Tris-base extender containing SPE (1, 5, 10 and 20 μg/mL) or without SPE (control). Diluted semen was cooled to 4 °C throughout one hour and frozen in 0.25 mL straws: prior to being stored in liquid nitrogen. Cryopresreved spermatozoa were assessed for post-thawing sperm motility, viability, acrosomal integrity, ultrastructure changes, antioxidant activities, lipid peroxidation and fertility rate. The current results clearly indicated that adding 10μg/mL SPE to the freezing extender significantly improved (P< 0.05) post-thawing motility and decrease the percentage of acrosomal damage (51.67±6.02% and 16.33±1.46%, respectively) compared with the control (28.33±4.41% and 26.33±1.77%, respectively). Moreover, addition of 10 μg/mL SPE to the semen extender significantly diminished (P< 0.05) MDA concentration (10.66±2.40 nmol/10⁹) compared with the control (22.66±4.26 nmol/10⁹). Therefore, the present results revealed that addition of 10μgl/mL SPE to the freezing extender might improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.     

Fertilization characteristics and in vitro embryo production with bovine sperm containing multiple nuclear vacuoles

An in vitro fertilization and culture system was used to determine the effect of multiple nuclear vacuoles in bovine spermatozoa on fertilization and early embryonic development. After swim-up, semen parameters were similar between 2 bulls except that 60% of spermatozoa from bull A contained multiple nuclear vacuoles, whereas no spermatozoa from bull B (control) contained vacuoles. In Experiment 1, in vitro–matured (IVM) oocytes were inseminated with frozen-thawed semen from the 2 bulls to determine the ability of vacuolated sperm to bind with the zona pellucida. The mean number of spermatozoa bound to the zona pellucida was less (P< 0.05) for bull A (85.7 ± 5.7; n = 112) than for bull B (108.9 ± 5.4; n = 130). In Experiment 2, the percentages of zonae penetrated by spermatozoa from bull A (151 of 201; 75%) and bull B (116 of 150; 77%) were not different. However, the percentages of vacuolated spermatozoa from bull A bound to (43%) and penetrating the zona pellucida (34%) were lower than those in the inseminate (60%). In Experiment 3, fertilization rates, as evidenced by the presence of two pronuclei, were not different for bull A (101 of 136; 74%) and bull B (89 of 115; 77%). In Experiment 4, there was no significant difference in percentage cleavage (72.1% versus 76%) and morulae (29.2% versus 34.8%) or blastocyst production (7.2% versus 8.4 %) for bulls A and B, respectively. Data suggest that spermatozoa with multiple nuclear vacuoles are defective in zona binding. However, vacuolated spermatozoa gaining access to the ooplasm apparantly participate in fertilization and early embryonic development. Mol. Reprod. Dev. 50:328–333, 1998. © 1998 Wiley-Liss, Inc.     

Double freezing of bovine semen

Fertility of bull spermatozoa cryopreserved in large volume by directional freezing technique, thawed, repackaged in straws and refrozen over liquid nitrogen vapor (double freezing, DF) was compared to conventional single freezing in straws (CF). Semen was collected from 6 bulls, 4 of which were selected for the field trial. Each semen collection was split into two parts, one frozen by CF and the other by DF. In vitro semen evaluations included motility (fresh, upon thawing and after 3 h incubation at 37 °C), viability and acrosome integrity. A total of 3610 cows and heifers were randomly inseminated by either CF or DF at about equal numbers. In vitro sperm analysis indicated no difference between CF and directional freezing in large volume and both were superior to DF (P < 0.001). Between-bull variations in fresh semen and in their reaction to CF or DF were apparent. Logistic regression analysis revealed that freezing method, bull, parity and inseminating technician, all had significant effect on pregnancy outcome (P ≤ 0.001 for all). Conception rate (CR) was 32.98% for CF and 28.05% for DF. Only in one bull conception rate by CF was significantly superior to DF (P < 0.05). When divided into heifers, primi- and pluriparous cows, only the difference in CR between the pluriparous cows was significant (P = 0.005). In conclusion, acceptable CR can be achieved by DF technique. These can be improved by selecting suitable bulls. The DF technique can be utilized in storage, sperm sexing and genome resource banking.     

Heterologous fertilization to characterize spermatozoa of the genus Bos

Advances in assisted reproductive techniques, specifically, development of protocols for production of in vitro matured, fertilized and cultured domestic bovine embryos, offer opportunities to apply these techniques to nondomestic bovidae in species preservation. Domestic bovine oocytes were inseminated with nondomestic bovine spermatozoa. Effects of heparin concentration, sperm concentration and their interaction on total and normal in vitro fertilization rates and on subsequent embryo development were evaluated. In different replications, semen from 3 Bos bison, 2 Bos gaurus, 1 Bos grunniens, and 1 Bos javanicus bulls was used. Treatment of spermatozoa included 2 heparin levels (2 and 8 micrograms/mL) and 3 sperm concentrations (1, 3 and 5 x 10(6)/mL). The B. grunniens bull exhibited excessive polyspermy in all treatments; therefore, 1 replicate was completed using 2 levels of heparin (0 and 1 microgram/mL) and 2 sperm concentrations (1 and 2 x 10(6)/mL). After 18 to 22 h, cumulus cells were removed from presumptive zygotes, and a portion thereof was compressed between a slide and coverslip and fixed in acetic acid:ethanol solution. Light microscopy was used to visualize pronuclei and the second polar body as a determinant of fertilization. Remaining presumptive zygotes were placed into embryo culture medium, and blastocyst development was assessed on Days 7 and 8 (fertilization = Day 0). Percentages of total and normal fertilization and of blastocyst formation were analyzed by a logistic regression model, isolating effects due to bull, heparin and sperm concentration, and to their interaction. Work presented here suggests that, just as in Bos taurus, the nondomestic bulls in the Bos species seem to have individual heparin and sperm concentration requirements for successful IVF. We conclude that each bull, domestic or nondomestic, needs to be evaluated individually. Preliminary sperm characterization using domestic cattle oocytes would result in a greater potential for generating purebred embryos of the desired species should scarce female gametes become available.     

Seasonal effects on circulating leptin in the lizard Sceloporus undulatus from two populations

Characterizing leptin's structure and function in mammals has been the subject of thousands of studies since 1994. Recently, the study of leptin has expanded to include its distribution in non-mammalian taxa, and the role that leptin plays in the reproductive axis. We demonstrated in a previous study that Sceloporus undulatus, fence lizards (ectotherms), express a leptin-like protein. In the current study we quantified seasonal variation in this putative leptin among free-ranging fence lizards from two populations characterized by early and late reproductive maturation (after one or two years, respectively). Immunoblots were performed on whole blood samples to detect leptin and estimate its titer. Leptin titers were higher in the reproductive population of S. undulatus (early maturing: 2.5 ± 0.2 μg/mL; late-maturing 2.2 ± 0.3 μg/mL; mean ± 2 S.E.), but both populations showed the same seasonal pattern. Leptin titers were lowest in fall when fat stores are expected to be highest (spring: 2.6 ± 0.3 μg/mL; summer: 2.6 ± 0.3; μg/mL; fall: 1.8 ± 0.3 μg/mL), consistent with findings of seasonal variation in free-ranging mammals. Our data support previous work asserting that lizards express leptin and that it has a similar physiological function in endotherms and ectotherms. Our long-term goal is to use leptin to manipulate age at maturity and to test fundamental questions in the evolution of life-history strategies.     

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30–36-month-old, 500–550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1–5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 ± 0.6 and 17.0 ± 0.8, respectively; mean ± S.E.M.) was higher (P < 0.05) in agreement with the incidence of nuclear vacuoles (14.0 ± 5.0 and 12.3 ± 2.3) and abnormal chromatin (24.4 ± 7.2 and 30.8 ± 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R² = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R² = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R² = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.     

Efficacy of a commercially available post-thaw bovine semen sexing kit in both single-ovulating and hyperstimulated cows

Currently, there are few inexpensive, reliable, effective methods for commercially separating X- and Y-chromosome bearing fresh and frozen bovine sperm. The objective of these experiments was to determine the efficacy of a commercially available post-thaw bovine semen sexing kit, HeiferPlus™ (HP) which claims to alter the sex ratio in favor of female calves following artificial insemination. Three trials included the insemination of hyperstimulated cows with Control or HP-treated semen, non-surgical embryo collection on Day 7, and a combined PCR/dot blot assay to determine embryo sex. Chi-square analysis showed that the Control group produced a greater proportion (p < 0.0005) of female embryos than the HP group. There were no differences in the proportions of transferable compared with degenerate embryos or in number of ovulations, embryos, and unfertilized ova collected from Control compared with HP groups. When treatments were combined, one of the two bulls used in the hyperstimulation studies produced an overall greater proportion of females (p < 0.05), suggesting a bull effect.Another trial involved the insemination of cows synchronized via OvSynch^® with fetal sexing via ultrasonography. Results of these studies indicated that HP semen sexing kit did not alter the sex ratio in favor of females in either hyperstimulated or single-ovulating cows; however, potential bull effects may be further evaluated to understand the capacity of HP with semen from specific bulls. Additionally, perhaps the sex of the surviving embryo can be manipulated by the maternal side, through ovarian, hormonal, oviductal, or uterine influences.     

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4 h at 5 °C, and at 0 and 2 h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4 ± 17.8 μm/sec) and high progressiveness (LIN: 65.1 ± 14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7 ± 25.6 μm/sec) but a nonprogressive trajectory (LIN: 33.1 ± 10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3 ± 24.3 μm/sec) and nonprogressive sperm (LIN: 39.6 ± 18.3%); and Subpopulation 4 included very rapid (VCL: 152.8 ± 25.7 μm/sec) and highly progressive sperm (LIN: 70.9 ± 13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P < 0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.     

Season of the year influences semen output and concentrations of testosterone in circulation of yaks (Poephagus grunniens L.)

Seasonal changes in plasma testosterone concentration and semen quality were evaluated in yak bulls throughout a 1-year period. Blood samples were collected every week from adult yak bulls (n = 15). These blood samples were analyzed for testosterone using a highly sensitive enzyme-linked immunoassay. Ejaculates were collected from five representative bulls each week. Ejaculate volume, progressive motility, live sperm count and sperm concentrations were determined. Mean testosterone in plasma was 1.03 ± 0.25 ng/ml. Concentrations of testosterone changed throughout the year (P < 0.05) and were found to be highest during the winter. It was also higher during the autumn than in summer and spring (P < 0.05). Mean ejaculate volume, progressive motility, live sperm count and spermatozoa concentration were 2.7 ± 0.3 ml, 72.8 ± 1.4%, 82.3 ± 0.9% and 968 ± 233 × 10⁶ ml⁻¹, respectively. Ejaculate volume and sperm concentration were higher (P < 0.05) in autumn than in other seasons. To conclude, a highly sensitive EIA for testosterone was developed and validated for yak plasma. Seasonal changes in semen quality were associated with changes in the concentration of testosterone in plasma from yak bulls.     

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

The use of in vitro fertilization techniques to investigate the fertilizing ability of bovine sperm with proximal cytoplasmic droplets

The objective of this experiment was to determine the effect of proximal droplets on sperm-oocyte binding, zona penetration, fertilization, and the developmental competence of oocytes fertilized by sperm with proximal droplets (PD) in an in vitro fertilization (IVF) and culture system. Frozen semen from three bulls (PD1, PD2 and PD3) with varying proportions of normal appearing sperm with proximal droplets and semen from a normal control bull (C) were used in this experiment. The mean number of sperm bound to the zona pellucida (26.8 +/- 2.0, n=100; 15.2 +/- 1.1, n=100; 16.2 +/- 1.0, n=100) for bulls PD1, PD2, and PD3, respectively, were significantly lower (P<0.05) than that of the control bull C (47.4+/- 1.9; n=114). No spermatozoa with PD were found bound to the zona pellucida and this finding was consistent among the three bulls. The percentage penetration of zonae for the bulls PD1, PD2 and PD3 (74%, 74/100; 71%, 71/100 and 69%, 69/100, respectively) were not different than that of bull C (72%, 179/245). Similarly, the mean number of sperm penetrating the zona pellucida (1.43+/- 1.2, 1.24 +/- 1.1 and 1.20 +/- 1.1, for bulls PD1, PD2 and PD3, respectively) were not different than that of bull C (1.45 +/- 1.1). However, fertilization rates (8.8%, 8/90; 16.8%, 16/95; and 10.6%, 11/103, for bulls PD1, PD2 and PD3, respectively) were lower (P<0.001) than that of bull C (68.7%; 77/112). Similarly, cleavage rates (5%, 10/200; 8%, 8/100 and 14%, 15/111) for the bulls PD1, PD2 and PD3, respectively, were lower than that of the control bull, C (60.7%; 79/130). Cleaved zygotes resulting from the fertilization of oocytes by apparently normal sperm from bulls with PD did not develop beyond cleavage, whereas, 43.8% (57/130) morulae and 20% (26/130) blastocysts were produced by oocytes fertilized by sperm from bull C. In summary, normal appearing sperm with PD did not bind to the zona pellucida. Apparently normal sperm with out proximal droplets co-existing in the semen along with sperm containing PD were also functionally deficient, resulting in reduced zonae binding and zygotes resulting from insemination with semen with a high percentage of PD did not develop beyond cleavage.     

Semen and reproductive profiles of genetically identical cloned bulls

In this comparative study, reproductive parameters and semen characteristics of cloned bulls (n = 3) derived from somatic cell nuclear transfer (SCNT) were compared to their original cell donor Holstein-Friesian (n = 2) bulls from the same enterprise to assess the differences in reproductive potential between a donor bull and its clones. The parameters evaluated included motility of fresh, frozen-thawed and Percoll-treated frozen-thawed spermatozoa, as well as in vitro fertilization (IVF) ability, embryo quality, birth and survival of calves following IVF and embryo transfer with frozen-thawed semen. With fresh semen, spermatozoa from one cloned bull had lower motility than its donor. Cloned bulls had higher velocity parameters in fresh semen, but those effects were not obvious in frozen-thawed or frozen-thawed semen selected with a Percoll gradient. Semen collected from cloned bulls had significantly higher IVF rates compared to donors; however, embryo development per cleaved embryo or quality of blastocysts did not differ between donors and cloned bulls. Pregnancy and live offspring rates from one donor and its cloned bull did not differ between fresh (40%, 16/40 versus 46%, 17/37) and vitrified/thawed (13%, 2/16 versus 25%, 4/16) embryo transfer following IVF. A total of 26 calves were obtained from genotypically identical donor and cloned bulls with no signs of phenotypical abnormalities. These preliminary results suggested that the physiology of surviving postpubertal cloned bulls and quality of collected semen had equivalent reproductive potential to their original cell donor, with no evidence of any deleterious effects in their progeny.     

Embryo development and structural analysis of in vitro matured bovine oocytes vitrified in flexipet denuding pipettes

The aim of this study was to compare the effectiveness of two different vitrification carrier systems for oocyte cryopreservation. In vitro matured (IVM) bovine oocytes were vitrified in open pulled straws (OPS) or flexipet denuding pipettes (FDP), and the effects of cryopreservation determined on the cytoskeletal components and developmental capacity of the oocytes. Three experimental groups were established according to whether the oocytes were vitrified in OPS (OPS group), FDP (FDP group) or left untreated (CTR group). Twenty two hours after the onset of maturation, sub-groups of 2–4 oocytes were pre-equilibrated in 1 mL of Hepes-TCM 199 with 20% fetal calf serum (FCS) (HM), 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 30 s. The oocytes were then transferred to a 20-μL drop of HM containing 20% DMSO, 20% EG and 0.5 M of sucrose, which was used to load the OPS or FDP before their immersion in liquid nitrogen (LN₂). Oocytes were thawed by plunging the OPS or FDP into 0.25 M sucrose in HM, and then placed for 5 min each in 0.15 and 0 M sucrose in HM. After warming, spindle configuration, chromosome distribution and embryo development were assessed. Frozen–thawed semen was used for fertilization. Zygotes were denuded at 22 h post-insemination, and cultured in SOF medium for 9 days at 38.5 °C in a 5% CO₂, 5% O₂ and 90% N₂ atmosphere. All experiments were performed using both cow and calf oocytes to establish sensitivity differences. After in vitro fertilization and culture, oocytes in the FDP group showed a lower cleavage rate than those in the OPS or control groups (P < 0.05), while blastocysts were only obtained in the OPS group, at a lower rate than controls. After warming, double fluorescent staining revealed higher rates of spindle and chromosome abnormalities in the FDP group compared to the OPS group (P < 0.05). No differences between cow and calf oocytes were observed in the different experiments. Our results indicate that the carrier system affects the capacity of IVM oocytes to survive cryopreservation. Unexpectedly, the flexipet denuding pipette failed to improve results and high rates of clustered chromatin and abnormal spindles were observed in calf and cow oocytes vitrified by the FDP method. In conclusion, the use of the flexipet denuding pipette modifies the cytoskeletal components and compromises the developmental capacity of in vitro matured calf and cow oocytes.     

Antioxidant activities of flavonol derivatives from the leaves and stem bark of Scutia buxifolia Reiss

This study evaluated the antioxidant activities in the leaves and stem bark fractions of Scutia buxifolia. Cerebral lipid peroxidation (TBARS) was induced by Fe(II) and radical-scavenging activity was determined by DPPH method. Folin–Ciocalteu was used to determine phenolic contents. Quercetin, quercitrin, isoquercitrin and rutin were isolated from leaf ethyl acetate fraction and their levels were measured by high performance liquid chromatography.-photodiode array detector.IC₅₀ (DPPH) varied from 4.35 ± 1.30 to 29.55 ± 0.54 μg/mL for stem bark and from 6.50 ± 0.40 to 30.54 ± 1.14 in the leaves. Ethyl acetate and butanolic fractions caused a sharp fall in TBARS production with IC₅₀ from 2.93 ± 2.17 to 40.46 ± 2.51 μg/mL for the leaves and 0.66 ± 0.17 to 27.3 ± 1.23 for the stem bark. Results obtained indicated that S. buxifolia has a great potential to prevent disease caused by the overproduction of free radicals and also it might be used as a potential source of natural antioxidant agents.     

Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen

Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n = 14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 μg/ml bovine pituitary extract, 5 μg/ml insulin, 10 ng/ml cholera toxin, and 50 μg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 μg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p < 0.05, n = 24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p < 0.05, n = 24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p < 0.05, n = 16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p < 0.05, n = 16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available.     

Strategies to improve fertility in Bos indicus postpubertal heifers and nonlactating cows submitted to fixed-time artificial insemination

Two experiments were designed to evaluate strategies to increase fertility of Bos indicus postpubertal heifers and nonlactating cows submitted to a fixed-time artificial insemination (TAI) protocol consisting of an intravaginal device containing 1.9 g of progesterone (CIDR) insertion + estradiol benzoate on Day 0, CIDR withdrawal + estradiol cypionate on Day 9, and TAI on Day 11. In Experiment 1, heifers (n = 1153) received a new or an 18-d previously used CIDR and, on Day 9, prostaglandin F_2α (PGF_2α) + 0, 200, or 300 IU equine chorionic gonadotropin (eCG). Heifers treated with a new CIDR had greater (least squares means ± SEM) serum concentration of progesterone on Day 9 (3.06 ± 0.09 ng/mL vs. 2.53 ± 0.09 ng/mL; P < 0.05) and a smaller follicle at TAI (11.61 ± 0.11 mm vs. 12.05 ± 0.12 mm; P < 0.05). Heifers with smaller follicles at TAI had lesser serum progesterone concentrations on Day 18 and reduced rates of ovulation, conception, and pregnancy (P < 0.05). Treatment with eCG improved (P < 0.05) follicle diameter at TAI (11.50 ± 0.10 mm, 11.90 ± 0.11 mm, and 12.00 ± 0.10 mm for 0, 100, and 200 IU, respectively), serum progesterone concentration on Day 18 (2.77 ± 0.11 ng/mL, 3.81 ± 0.11 ng/mL, and 4.87 ± 0.11 ng/mL), and rates of ovulation (83.8%, 88.5%, and 94.3%) and pregnancy (41.3%, 47.0%, and 46.7%). In Experiment 2, nonlactating Nelore cows (n = 702) received PGF_2α treatment on Days 7 or 9 and, on Day 9, 0 or 300 IU eCG. Cows receiving PGF_2α on Day 7 had lesser serum progesterone concentrations on Day 9 (3.05 ± 0.21 ng/mL vs. 4.58 ± 0.21 ng/mL; P < 0.05), a larger follicle at TAI (11.54 ± 0.21 mm vs. 10.84 ± 0.21 mm; P < 0.05), and improved (P < 0.05) rates of ovulation (85.4% vs. 77.0%), conception (60.9% vs. 47.2%), and pregnancy (52.0% vs. 36.4%). Treatment with eCG improved (P < 0.05) serum progesterone concentration on Day 18 (3.24 ± 0.14 ng/mL vs. 4.55 ± 0.14 ng/mL) and the rates of ovulation (72.4% vs. 90.0%) and pregnancy (37.5% vs. 50.8%). In conclusion, giving PGF_2α earlier in the protocol in nonlactating cows and eCG treatment in postpubertal heifers and nonlactating cows improved fertility in response to a TAI (progesterone + estradiol) protocol.