Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls

The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (P<0.05), while at 18 hpi, the proportion of the three PN stages was similar to those of bulls I and IV, bull II being delayed. In a second experiment, the kinetics of in vitro embryo development was compared using time-lapse cinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex ratio of the embryos. Moreover, those parameters might be related.     

Effect of reducing sperm concentration during IVF on the ability to distinguish between bulls of high and low field fertility: work in progress

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5 x 10(6)sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125 x 10(6)sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5 x 10(6)sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5 x 10(6)sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.     

Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially?

It has been reported that the mammalian female could have a preconceptual influence on the sex of her offspring, and it has been hypothesized that this influence could go some way toward accounting for the reported lower fertility following insemination with sex-sorted sperm. To test whether in vitro matured oocytes are able to select X- or Y-bearing spermatozoa following in vitro fertilization (IVF), we fertilized in vitro 1788 oocytes with X-sorted semen, Y-sorted semen, a mix of X- and Y-sorted semen, and unsorted semen from the same bull, and cultured until Day 9. Fertility was assessed by recording cleavage rate at 48 h postinsemination (hpi) and blastocyst development until Day 9. Embryos were sexed at the two- to four-cell stage and the blastocyst stage. The proportion of zygotes cleaving at 48 hpi was not different between X- and Y-sorted groups and the mix of X- and Y-sorted semen group; however, all were significantly lower than the unsorted group (P < 0.001). Blastocyst yield on Day 6 was significantly higher (P < or = 0.01) in the control group compared with the rest of the groups. Cumulative blastocyst yields on Days 7, 8, and 9 were also significantly higher (P < or = 0.01) in the unsorted group compared with the sorted groups. The proportion of female and male two- to four-cell embryos obtained following IVF with X- and Y-sorted sperm was 88% and 89%, respectively and the sex ratio at the two- to four-cell stage was not different following IVF with unsorted or sorted/recombined sperm (56.9% males vs. 57% males, respectively). At the blastocyst stage, similar percentages were obtained. In conclusion, the differences in cleavage and blastocyst development using sorted versus unsorted sperm are not due to the oocyte preferentially selecting sperm of one sex over another, but are more likely due to spermatic damage caused by the sorting procedure.     

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.     

Seasonal variation in semen quality in Bos indicus and Bos taurus bulls raised under tropical conditions

In the present study, we tested the hypothesis that Bos taurus taurus bulls have greater reactive oxygen species (ROS) and lower activity of antioxidant enzymes in their semen than Bos taurus indicus bulls. Sixteen Simmental bulls (B. t. taurus) and 11 Nelore bulls (B. t. indicus) were managed extensively in a tropical environment. Semen was collected twice annually (summer and winter) for 2 consecutive years. Simmental bulls had significantly higher percentages of major sperm defects during the summer than the winter (20.3+/-3.1% versus 12.2+/-2.4%, respectively; mean+/-S.E.M.). There was an interaction of breed and season for minor sperm defects (P=0.037; highest in Nelore bulls in the summer) and an effect of season on total defects (P=0.066; higher in summer). To evaluate oxidative damage, malondialdehyde (lipid-peroxidation metabolite) concentrations were indirectly measured by semen concentrations of thiobarbituric acid reactive substances (TBARS); these were higher in summer than in winter (728.1+/-79.3ng/mL versus 423.8+/-72.6ng/mL, respectively; P=0.01). Glutathione peroxidase/redutase (GPx) activity in semen was higher in Simmental versus Nelore bulls (741.6+/-62.1 versus 510.2+/-62.8; P<0.01). However, superoxide dismutase (SOD), another antioxidant enzyme, was not significantly affected by breed or season. There were correlations between TBARS and sperm primary defects during the summer for both Simmental and Nelore bulls (r=0.59, P=0.021 and r=0.40, P=0.034, respectively), and between SOD and primary defects during summer for Simmental bulls only (r=-0.51, P=0.041). In conclusion, there was a higher level of lipid peroxidation (ROS) in semen of Simmental versus Nelore bulls; apparently the higher GPx activity in Simmental bulls was insufficient to avoid damage that occurred concurrent with increased ROS production during the summer.     

The effect of gamete co-incubation time during in vitro fertilization with frozen-thawed unsorted and sex-sorted ram spermatozoa on the development of in vitro matured adult and prepubertal ewe oocytes

In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubated with unsorted or sex-sorted frozen-thawed spermatozoa for 2 to 3 h (short) or 18 to 20 h (long) to determine the effects of reducing the gamete co-incubation time during IVF on subsequent embryonic development in vitro. For oocytes derived from adult ewes, there were no differences in oocyte fertilization and cleavage at 24 h post insemination (hpi) between types of spermatozoa or co-incubation times (P > 0.05). By 48 hpi, oocyte cleavage was higher after a short (390/602, 64.8%) compared with a long (381/617, 61.7%) co-incubation (P < 0.05), and was not significantly different for unsorted (266/372, 71.5%) and sex-sorted (505/849, 59.9%) spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (150/266, 56.4%) and sex-sorted (295/505, 58.4%) spermatozoa, but was higher after a short (240/390, 61.5%) than long (205/381, 53.8%) co-incubation (P < 0.05). Oocyte development to the blastocyst stage was not different for unsorted (150/372; 40.3%) and sex-sorted (295/847; 34.8%) spermatozoa but was significantly increased by a short (240/602, 39.9%) compared with a long (205/617, 33.2%) co-incubation. Fertilization of oocytes from prepubertal ewes was similar for types of spermatozoa and for duration of co-incubation. Oocyte cleavage (48 hpi) was similar for a short (241/377, 63.9%) and long (226/349, 64.8%) co-incubation with unsorted spermatozoa, but was increased (P < 0.05) by a long co-incubation (286/500, 57.2% versus 163/517, 31.5%) with sex-sorted spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (230/467, 49.3%) and sex-sorted (186/449, 41.4%) spermatozoa, and a short (200/404, 49.5%) or long (216/512, 42.1%) co-incubation. However, oocyte development to the blastocyst stage was higher (P < 0.05) after IVF with unsorted (230/726, 37.1%) than sex-sorted (186/1017, 18.3%) spermatozoa. Reducing the duration of gamete co-incubation did not deleteriously affect the in vitro development of adult and prepubertal ewe derived oocytes after IVF with unsorted and sex-sorted spermatozoa. In general, sex-sorting had no substantial influence on fertilization and embryo development rates.     

Dietary protein intake impacts human skeletal muscle protein fractional synthetic rates after endurance exercise

This investigation evaluated the physiological impact of different dietary protein intakes on skeletal muscle protein synthesis postexercise in endurance runners. Five endurance-trained, male runners participated in a randomized, crossover design diet intervention, where they consumed either a low (0.8 g/kg; LP)-, moderate (1.8 g/kg; MP)-, or high (3.6 g/kg; HP)-protein diet for 4 wk. Diets were designed to be eucaloric with carbohydrate, fat, and protein approximating 60, 30, and 10%; 55, 30, and 15%; and 40, 30, and 30% for LP, MP, and HP, respectively. Substrate oxidation was assessed via indirect calorimetry at 3 wk of the dietary interventions. Mixed-muscle protein fractional synthetic rate (FSR) was measured after an endurance run (75 min at 70% V(O2 peak)) using a primed, continuous infusion of [(2)H(5)]phenylalanine. Protein oxidation increased with increasing protein intake, with each trial being significantly different from the other (P < 0.01). FSR after exercise was significantly greater for LP (0.083%/h) and MP (0.078%/h) than for HP (0.052%/h; P < 0.05). There was no difference in FSR between LP and MP. This is the first investigation to establish that habitual dietary protein intake in humans modulates skeletal muscle protein synthesis after an endurance exercise bout. Future studies directed at mechanisms by which level of protein intake influences skeletal muscle turnover are needed.     

Evaluation of fertility status of Murrah buffalo bulls in vitro using zona-free hamster eggs

Cryopreserved semen samples from 10 Murrah buffalo bulls were used for sperm penetration bioassay using zona-free hamster oocytes. The samples were evaluated for sperm motility, viability and acrosome integrity. Actively motile spermatozoa recovered by the swim-up technique were capacitated using calcium ionophore A(2 3 1 8 7). Mature female golden hamsters were superovulated with 50 IU PMSG followed 56 h later by 75 IU hCG. Cumulus mass, recovered by puncture of oviducts at the infundibulum region, was treated with 0.1% hyaluronidase and 0.1% trypsin to obtain zona-free oocytes. After coincubation of zona-free oocytes with capacitated buffalo spermatozoa, scoring was done as fertilization percentage and fertilization index. The correlation coefficients with conception rate were statistically significant with fertilization percentage (r = 0.588, P < 0.05) and fertilization index (r = 0.660, P < 0.01). However, conventional parameters like viability, motility and acrosome integrity showed poor correlation with conception rate.     

Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen-thawed buffalo semen

Frozen-thawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gradient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm (%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm separation methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was compared in experiment II. Swim-up separated sperm showed a higher motility (P<0.05), while percent recovery of motile sperm was higher with Percoll separation (P<0.05). Membrane integrity (%) of sperm separated with swim-up was significantly higher (P<0.05) as compared to sperm separated with Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index (P<0.001). Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleavage index (P<0.05), while the Percoll gradient method did not. It has been concluded that separation of sperm from frozen-thawed buffalo semen by swim-up method can be more expedient for IVF in buffalo.     

Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: efficiency and kinetics of oocyte fertilization and embryo development

The present experiments were designed to study the effects of Robertsonian translocations on the efficiency and kinetics of in vitro fertilization and early and advanced embryo development. Spermatozoa from bulls with rob(16;20), rob(1;29) and normal karyotype (A, B and C, respectively) were used. Oocytes were matured, fertilized and cultured by the standard protocol described previously. Twenty-four hours after fertilization, adequate numbers of oocytes were fixed, stained and examined. The development of embryos was evaluated on days 2 (D2), 7 (D7) and 8 (D8) after fertilization. The rate of normally fertilized oocytes was significantly lower (p < or = 0.01) for bull A than for bulls B and C. However, no significant differences in the kinetics of fertilization were found between bulls A, B and C. The D2 cleavage rate of embryos was significantly lower (p < or = 0.01) for bull A than for bulls B and C. Both D7 and D8 blastocyst rates for bull A or bull B were significantly lower (p < or = 0.01 or p < or = 0.05) than those for bull C. The percentages of both D7 advanced blastocysts and D8 expanded blastocysts were significantly lower (p < or = 0.01) for bulls A and B than for bull C. In conclusion, for rob(16;20), the efficiency of fertilization was strongly reduced; it resulted in low early and advanced embryo development. On the other hand, for the rob(1;29), neither fertilization nor early embryo development were affected and only advanced embryo development was decreased. But for both translocations, blastocyst formation was significantly delayed.     

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.     

Metabolic and growth response of mink (Neovison vison) kits until 10 weeks of age when exposed to different dietary protein provision

Growth performance and metabolism were investigated in mink kits (n = 210) exposed to the same dietary treatment as their dams (n = 30), i.e. high (HP; 61% of metabolisable energy, ME), medium (MP; 48% of ME) or low (LP; 30% of ME) protein supply, from birth until 10 weeks of age. The kits were weighed weekly, and were measured by means of balance experiment and indirect calorimetry, in weeks eight and nine post-partum (p.p.). At weaning (seven weeks p.p.) and 10 weeks p.p. one kit per litter was killed and blood, liver and kidneys were collected. Plasma amino acid profiles, and hepatic abundance of mRNA for phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-biphosphatase, pyruvate kinase and glucose-6-phosphatase (G-6-Pase) by q-PCR, were determined. There were no differences in live weights among kits the first four weeks of life when kits solely consumed milk, but male LP kits were the heaviest. After transition to solid feed MP kits weighed most at nine weeks of age (p < 0.05). At eight weeks of age, the kits fed the LP diet retained less (p < 0.05) N than HP and MP kits. Heat production did not differ among kits, although protein oxidation was higher (p < 0.001) in HP kits than in LP kits. Kits fed the LP diet had lower (p < 0.05) plasma concentrations of lysine, methionine and leucine than MP kits. Dietary treatment was not reflected in the relative abundance of any of the studied mRNAs, but kits had significantly lower abundance of all studied mRNA than their dams, ranging from 83% less PEPCK abundance to 40% less for G-6-Pase. The kidney mass was smallest (p < 0.01) in kits fed the LP diet, and liver masses were largest (p < 0.001) in HP kits. The results indicate that the LP diet did not meet the protein requirements for mink kits in the transition period from milk to solid feed. The capacity to regulate the rate of gluconeogenesis was even more limited in young mink kits than in adult dams. However, young mink kits can regulate protein oxidation in response to dietary protein supply, probably by adapting the size of the liver and kidneys to the level of protein supply.     

Pentoxifylline improves in vitro fertilization and subsequent development of bovine oocytes

The purpose of this study was to examine whether pentoxifylline improves in vitro fertilization and developmental rates of bovine oocytes. In the first experiment, we examined the effects on the fertilization rate of various concentrations of pentoxifylline (0-7.5 mM) combined with heparin (10 IU/mL). In the second experiment, we examined fertilization cleavage and blastocyst rates after frozen-thawed spermatozoa, obtained from four different bulls, were incubated with heparin (10 IU/mL) with or without caffeine (5 mM) or pentoxifylline (5 mM). In the first experiment, a significantly higher fertilization rate was obtained in heparin containing 5 mM pentoxifylline compared to that in heparin alone or in heparin containing 7.5 mM pentoxifylline (86% vs 60% vs 64%, respectively). The percentage of monospermy in 5 mM pentoxifylline (81%) was significantly higher than in heparin alone (57%). In the second experiment, the interactions among Bulls A, B, C, and D; between treatments (pentoxifylline-with-heparin, caffeine-with-heparin and heparin alone), and between bulls and treatments were analyzed for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts. Among bulls, there was a significant difference in the number of oocytes penetrated (P < 0.01), monospermic oocytes (P < 0.05), cleaved oocytes (P < 0.001), and blastocysts (P < 0.001). Between treatments, there was a significant difference in the number of oocytes penetrated (P < 0.001), monospermic oocytes (P < 0.01) and cleaved oocytes (P < 0.001). Interaction between bulls and treatments was observed for the number of oocytes penetrated (P < 0.05). Individually, for Bulls A, C and D, the numbers of oocytes penetrated and monospermic oocytes in pentoxifylline-with-heparin were significantly higher than in heparin alone. For Bull D, significantly higher results were obtained for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts in pentoxifylline-with-heparin compared to caffeine-with-heparin and heparin alone (P < 0.05). These results suggest that treating sperm with 5 mM pentoxifylline in combination with heparin is effective for bovine in vitro fertilization and it that this treatment is effective even for bulls that produce low fertilization and blastocysts after sperm treatment with caffeine-with-heparin or heparin alone.     

Effects of two treatments on semen from different bulls on in vitro fertilization results of bovine oocytes

The semen of six different bulls was used to examine the effects of treatment with caffeine or caffeine plus Ca-ionophre on in vitro fertilization, cleavage and development into morulae of in vitro matured bovine oocytes. In vitro fertilization results (formation of both pronuclei, cleavage and development to >/= four-cell stage were significantly (P<0.01) higher using caffeine plus Ca-ionophre than those using only caffeine. The rates of fertilization and first cleavage were only slightly variable among the bulls. However, the present data showed significant variability in formation of both pronuclei (36 to 75%) of fertilized ova and development to the >/=4cell stage (39 to 71%) by different bulls. Development into morulae of ova recovered from the rabbit oviduct did not show any significant differences in relation to sperm treatments or individual bulls.     

Paternal influence on the time of first embryonic cleavage post insemination and the implications for subsequent bovine embryo development in vitro and fertility in vivo

The objectives of this study were: (1) to evaluate the effect of sire on the time from insemination to first cleavage following insemination in vitro and the relationship of this parameter to field fertility and (2) to establish the relationship between the kinetics of cleavage in vitro and oocyte developmental competence for bulls of known field fertility. Frozen semen from six bulls with 150-day non-return rates ranging from 57-78% was used. In experiment 1, after insemination with semen from one of the six bulls, presumptive zygotes were transferred to IVC in droplets of synthetic oviduct fluid. Droplets were examined at 24, 27, 30, 33, 36, 42, and 48 hr after insemination and the number of cleaved oocytes was recorded. Blastocyst yield was recorded on Days 6-, 7-, and 8-post insemination. In experiment 2, culture droplets were examined at 30, 36, and 48 hr after insemination. At each time point, the number of cleaved embryos was recorded and these embryos were transferred into new droplets and were cultured separately for the duration of the experiment. The proportion of embryos developing to the blastocyst stage was recorded for each of the groups for each bull. The best predictor of field fertility was a model containing 33-hpi-cleavage percentage only (r = 0.689, P < 0.0001). There was also a significant correlation between blastocyst yield and non-return rate, with Day 7 blastocyst yield having the highest correlation (r = 0.356), although this was relatively low in comparison. In experiment 2, irrespective of sire, a significantly higher proportion of those early-cleaving oocytes (before 30 hpi) developed to blastocysts than those cleaving later. In most cases, a higher proportion of blastocysts derived from early-cleaving oocytes hatched from the zona pellucida suggesting that such blastocysts are of superior quality to those derived from late-cleaving oocytes. In conclusion these data confirm our earlier observations that earliest cleaving zygotes are more competent in terms of development to the blastocyst stage than those that cleave later. This phenomenon is independent of the sire used. However, we have demonstrated that the kinetics of early embryonic development as measured by the timing of the first cleavage division post insemination vary between different bulls and that these differences can be used to discriminate between bulls of high and low bull field fertility.     

Evaluation of sperm subpopulation structure in relation to in vitro sperm–oocyte interaction of frozen-thawed semen from Holstein bulls

The present study examined the relationship between the relative amount of high motile sperm and sperm–oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.     

Relationship between bull field fertility and in vitro embryo production using sperm preparation methods with and without somatic cell co-culture

Experiments were designed to compare rates of embryonic development following oocyte exposure to cryopreserved spermatozoa from bulls of varying proven fertility, utilizing 3 different sperm preparation methods prior to oocyte introduction. These included 1) sperm co-culture with bovine oviductal epithelial cells (BOEC); 2) sperm co-culture with buffalo rat liver cells (BRLC); or 3) control culture in a routine, cell-free culture system. Semen from 9 bulls was classified by lifetime 60- to 90-d nonreturn rates as having either (mean +/- SEM) high (n=3) 73.2 +/- 3a, medium (n=3) 70.3 +/- 2b or low (n=3) 65.8 +/- 3c field fertility ((ac)p< 0.01; (bc)p< 0.05). There was no difference in embryo cleavage rates for spermatozoa from the high (58 +/- 18%), medium (57 +/-23%) or low (57 +/- 18%) fertility groups. Development to morula or beyond of oocytes fertilized with high (53 +/- 30%) or low (58 +/- 27%) fertility semen tended (P<0.10) to be higher than of those fertilized with medium fertility (33 +/- 28%) semen. This lack of relationship between in vivo fertility and in vitro embryo outcome was consistent across all sperm preparation methods. Therefore, pooled data were used to evaluate the effect of sperm preparation on embryo outcome. There was no difference in embryo cleavage rates between BOEC monolayers (51 +/- 22%), BRLC monolayers (60 +/- 20%) and the cell-free controls (60 +/- 17%). Subsequent embryonic development to compact morula and beyond was higher (P<0.01) with the BRLC monolayer treatment (61 +/- 28%) than with the BOEC monolayers (42 +/- 33%) or control culture (39 +/- 24%). In conclusion, these studies suggest that there is no predictive relationship between bull field fertility (in the ranges evaluated here) and in vitro embryo cleavage or development rates. However, oocytes inseminated with sperm cells co-cultured on BRLC monolayers develop to the morula stage or beyond at a higher rate than oocytes inseminated with spermatozoa from the BOEC or cell-free system.     

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.     

A potential relationship between the acrosome response characteristics of bovine spermatozoa and their in vitro fertilizing ability

The aim of the work was to study a potential relationship between acrosome response characteristics of bovine spermatozoa and their ability to fertilize oocytes and produce in vitro embryos. Sperm of artificial insemination bulls with a high rate (22.0 +/- 4.1%, group A, n = 7) or a low rate (10.3 +/- 4.1%, group B, n = 8) of embryos were used. For acrosome assessment, motile spermatozoa from a Percoll gradient were incubated with or without heparin and examined by the fix-vital sperm assay (FVSA). The differences between the heparin-treated (H+) and the non-treated (H-) spermatozoa were significant (p < 0.01) in all bulls at all tested intervals. According to the kinetics of the heparin response, the bulls fell into three categories: fast (FR, n = 7), moderate (MR, n = 5) or slow (SR, n = 3) acrosome responses (p < 0.01). Five MR bulls were found in group A in comparison with two MR bulls in group B (57.1 vs 12.5%; p < 0.05). Intensity of the acrosome response (response index) was significantly higher in bull group A compared with bull group B (7.0 vs 4.6, p < 0.01). A positive correlation was recorded between response index and embryo rate (r = 0.668, p < 0.01). In conclusion (a) the kinetics of spermatozoa response to heparin may be important for in vitro fertilization, bulls with a moderate response appearing to be most suitable for embryo production; (b) greater spermatozoa response to heparin was related to more effective embryo production.     

Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture

Frozen-thawed spermatozoa obtained from six different bulls were suspended in Brackett and Oliphant's (BO) medium (14), with or without 10 mM caffeine, after washing. A 50-mul aliquot of the sperm suspension was added to the 50-mul BO medium supplemented with bovine serum albumin (BSA, 20 mg/ml) and heparin (20 mug/ml) in which the bovine follicular oocytes matured in culture had been introduced previously. The proportion (35%) of oocytes penetrated in the presence of heparin alone 20 to 24 h after insemination was not significantly different from those (32%) penetrated in the presence of caffeine alone as reported previously (1). When heparin was added to the caffeine in the fertilization medium, the penetration rate of oocytes increased significantly to 68% (P < 0.001), indicating that both chemicals act sinergistically to induce capacitation and/or acrosome reaction of spermatozoa and stimulate in vitro fertilization of cattle oocytes. However, great variation in penetration rates (35 to 96%) was observed among the different bulls. The optimal concentration of heparin in the suspension medium in which the highest rate of oocyte penetration took place was 10 mug/ml.