Growth performance and fertility of Windsnyer boars supplemented with α-tocopherol.

The purpose of the present work was to determine the response in growth performance and spermatozoa characteristics of Windsnyer boars supplemented with progressive levels of α-tocopherol. Twenty Windsnyer boars aged 12 weeks with an average body weight of 19.5 ± 2.67 kg were used. Each boar was housed individually in a 1.54 × 0.8 m pen in environmentally controlled house with the temperature ranging from 22 to 25 °C. Five boars were randomly assigned to each diet containing 0, 40, 70 and 90 IU of α-tocopherol. The growth performance experiment lasted for 12 weeks. Subsequently, boars were humanely slaughtered for analyses of testicular development and spermatozoa characteristics. Polynomial regression was used to analyse data. There was a linear response (P < 0.05) in average daily gain and feed conversion ratio as α-tocopherol levels increased. Left and right testicular weights showed a linear increase (P < 0.05) with increasing levels of α-tocopherol. Weights of left and right epididymis exhibited quadratic response (P < 0.05). Seminiferous tube area responded in a quadratic fashion (P < 0.05). There was a quadratic relationship (P < 0.05) between semen volume, straight-line velocity and live spermatozoa. Dead spermatozoa and head abnormalities exhibited linear decrease (P < 0.05). In conclusion, inclusion of α-tocopherol improved growth performance and fertility of Windsnyer boars.

Significant heparin effect on bovine embryo development during sexed in vitro fertilization.

The aim of this study was to investigate the effect of different heparin concentrations in the course of sexed in vitro fertilization (IVF), on bovine embryonic development and development to term following embryo transfer (ET). With a total of 9156 oocytes for IVF, sorted as well as unsorted sperm from four bulls had different heparin requirements for achieving the highest rate of development in vitro. However, when optimal heparin concentrations were used (40 to 80 µg/ml), the performance of X-sorted sperm (0.3 × 106/ml/IVF droplet) from all four bulls, as judged by blastocyst development (Bulls A, B, C, and D: 25.2, 19.7, 25.1, and 9.8%, respectively), was significantly increased, and the blastocyst rate was comparable to that observed with unsorted sperm at certain heparin concentrations within the four bulls. We determined that near-optimal blastocyst development was possible with sorted sperm from all four bulls, when a heparin concentration of 40 µg/ml was used. Pregnancy rates at d 70 post ET ranged from 39.1 to 40.3% (P > 0.05), and the calving rates ranged from 34.4 to 35.1% (P > 0.05), when heparin was used at a concentration of 10 µg/ml (n = 236), 20 µg/ml (n = 189), and 40 µg/ml (n = 305), respectively. Our study demonstrates that, although the sorted sperm of different bulls performed optimally over a range of heparin concentrations, a generally accepted heparin concentration of 40 µg/ml can be set for sexed IVF. This improvement is beneficial when sexed embryo production by ovum pickup and IVF is an essential component of genetic breeding programs.

Comparison of four different permitting and combination of two best cryoprotectants on freezing Nguni sperm evaluated with the aid of computer aided sperm analysis.

Cryopreservation has been reported to damage approximately 40-50% of viable sperm in bull semen. The present study was undertaken to assess the cryo-effectiveness of glycerol (GLY), ethylene glycol (EG), dimethyl sulfoxide (DMSO) and propylene glycol (PND) as cryoprotectant during the cryopreservation of Nguni bull semen. Semen was collected from 18 Nguni bulls and evaluated macroscopically and microscopically for sperm parameters. Thereafter, the semen samples were diluted with egg-yolk citrate extender supplemented with either 12% GLY or DMSO or EG or PND cryoprotectant. Semen samples were loaded into straws and placed into a controlled rate programmable freezer and stored in a liquid nitrogen tank. Following semen thawing, artificial insemination (AI) was done on synchronized Nguni cows. The in vitro fertilization (IVF) was conducted on cow's oocytes to test the fertilizing ability. Data was analyzed with the aid of ANOVA. A significant difference (p < 0.05) was recorded between fresh total sperm motility rate (94.7 ± 2.6%) and frozen-thawed sperm total motility rate with GLY (77.8 ± 11.0%), EG (20.4 ± 10.1%), DMSO (15.7 ± 11.9%) and PND (11.2 ± 11.3%). Interestingly, a positive correlation between total sperm motility and pregnancy rate (r = 0.42) was recorded. However, a negative correlation of Nguni sperm parameters with IVF (r = -0.53) was obtained. The freezing-thawing process did reduce the Nguni sperm total sperm motility percentage.

Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture.

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.

Magnesium is a critical element for competent development of bovine embryos.

The study was designed to determine the impact of magnesium (Mg2+) on bovine embryo development. We found that two commercially available sources of bovine serum albumin (BSA) and fetal bovine serum (FBS) contained different amounts of Mg2+ residue: 4 ppm in ICPbio BSA, 114 ppm in Sigma BSA, and 44 ppm in FBS. When CR1 was used as basal medium, PVA and ICPbio BSA produced the lowest blastocyst yield (2.2-2.3%), whereas Sigma BSA increased blastocyst yield to 18.9% (P < 0.05). Supplementation of 1.4 mM MgCl2 into the medium increased the blastocyst rate in the ICPbio BSA group (29.4%) but not in the PVA group (5.4%; P < 0.05) to a level comparable to that of the FBS group (33.7%; P > 0.05). We next found that increasing concentrations of MgCl2 in the culture medium (ICPbio BSA) elevated blastocyst rate from 2.6% (0 mM), 38.4% (0.35 mM) to 50.2% (1.4 mM; P < 0.05), further maintained at 44.9% (2.1 mM) and 43.4% (2.8 mM) (P > 0.05). However, blastocyst rate was reduced to 31.4% (4.2 mM) and 29.4% (5.6 mM) when MgCl2 supplement was increased (P < 0.05). Comparable blastocyst development was achieved in both ICPbio BSA (30.0-33.1%) and Sigma BSA (37.4-38.7%) groups when 1.4 mM Mg2+ was supplemented regardless of its source (MgCl2 vs. MgSO4; P > 0.05). In embryo transfer experiments, higher rates of pregnancy (54.3 vs. 41.5%) and calving (44.3 vs. 32.5%) were achieved in the CR1-Mg2+-supplemented BSA group compared with the FBS group with co-culture, respectively (P < 0.05). These results demonstrate that Mg2+ is a key ion that promotes competent blastocyst and term development. Therefore, a simple and efficient defined medium (CR1-Mg2+-BSA) can successfully replace complex serum and somatic cell co-culture.

Bovine blastocyst development in vitro: timing, sex, and viability following vitrification.

Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.