Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soy bean extract.

Semen extenders containing components such as egg yolk and skim milk are difficult to standardize and they introduce the risk of microbial contamination. A well-defined extender not originating from animal tissues would present a valuable contribution to the AI industry. We evaluated the fertility of bovine semen cryopreserved with 3 different extenders: 1) TRIS-Standard, prepared at 2 local AI laboratories, containing 20% (v/v) pasteurized egg yolk, 2) TRIS-Concentrate, prepared by adding 20% (v/v) pasteurized egg yolk and 1:5 (v/v) nonpyrogenic water, and 3) Biociphos Plus, a soybean extract containing extender, prepared by adding 1:5 nonpyrogenic water. Ejaculates of 4 Holstein bulls were split into 3 aliquots and cryopreserved with the 3 extenders. Prior to this study, the semen dose-response curve for each of the 4 bulls was developed in a field trial by freezing the semen and randomly distributing the straws throughout the Netherlands for insemination. Optimal semen doses were thus established to detect the effect of extenders on fertility, evaluated by 56-day non-return rate (NR56), and by the estimated conception rate and the calving rate, given a conception. We used the multiphasic model developed by Grossman et al. (7). A total of 22,246 first and second inseminations were recorded. The NR56 ranged among bulls from 67.0 to 70.1% for Tris-Standard, from 67.5 to 69.9% for Tris-Concentrate and from 60.2 to 66.7% for Biociphos Plus. No significant differences in NR56 were detected between Tris-Standard and Tris-Concentrate (P=0.54), whereas Biociphos Plus resulted in a significantly lower NR56 than Tris-Standard and Tris-Concentrate (P<0.05). Estimated conception rate was 72.1, 73.6 and 69.6% and estimated calving rate, given a conception was 80.6, 78.3 and 77.1 for Tris-Standard, Tris-Concentrate and Biociphos Plus, respectively. These results indicate that 1) semen extended with a custom made TRIS-Concentrate can be succesfully used in the field resulting in comparable fertility with Tris-Standard; 2) semen extended with Biociphos Plus results in a significant reduction in the NR56; 3) extender source may affect both conception rate and calving rate, given a conception, i.e., extrinsic and intrinsic sperm factors (4).

Embryonic origins of health: long-term effects of IVF in human and livestock.

Assisted reproduction technologies have been introduced 1) to overcome reproductive failures in the human, 2) to increase the number of offspring from selected females and 3) to reduce generation intervals in livestock in farm animals. The successful introduction of these technologies in clinical practice and in livestock breeding programs is the result of enormous scientific efforts. In general, the offspring generated by IVF (human) and IVP (cattle) is normal, but as numbers increase the restraints and drawbacks of new reproductive technologies become visible with respect to the overall efficiency as well as to the occurrence of abnormalities and/or anomalies in the offspring. The objective of the present symposium on "Embryonic Origins of Health" is to present "the-state-of-the-art" and to discuss the restraints and possible long term effects of the application of assisted reproduction technology in both human and livestock. This introduction to the symposium focuses on the relation between early embryonic development and post-natal health. We hypothesize that IVF in the human and IVP in cattle influence the timing of cell-cell interactions during the early stages of embryogenesis which finally result in a incorrect timing of gene expression during the phylotypic stage and subsequent organogenesis. These deviations in embryonic timing might have consequence for the postnatal homeostasis and health.

Effects of different reproduction techniques: AI MOET or IVP, on health and welfare of bovine offspring.

Since the introduction of in vitro production (IVP) of bovine and sheep pre-implantation embryos, increased birth weights and other deviations of IVP calves and lambs compared with AI or MOET offspring have been reported. Study 1 of the present paper, a comparison between AI, MOET and IVP (co-culture/serum) calves with respect to calf and calving characteristics in large-scale field conditions, confirms these reports. In addition, it is shown that MOET calves tend towards higher birth weights and have significantly longer gestations and more difficult calvings than AI calves. It is presently unknown if the effect of IVP is also observed later in life. In this paper, data on reproduction characteristics of bovine IVP co-culture/serum offspring are presented. Semen production--and non return data of one year old IVP bulls and superovulation-, AI- and OPU/IVP results of one year old IVP heifers are compared with those of one year old AI and MOET animals producing semen or embryos in the same time period. So far, there are no indications that the use of IVP is reflected in deviate reproduction characteristics of bovine IVP offspring. It has been suggested that use of co-culture cells and serum during in vitro culture of bovine (and sheep) embryos may partially explain the increased birth weights and other deviations of bovine and sheep IVP offspring. Deletion of these factors in semi-defined culture media, e.g. Synthetic Oviductal Fluid (SOF), could result in more normal offspring. Study 2 investigates this hypothesis in both field conditions (Study 2a, comparing AI, IVP co-culture/serum and IVP SOF calves) and in semi-standardized conditions (Study 2b, comparing MOET, IVP co-culture/serum and IVP SOF calves at one herd). In Study 2a, although IVP SOF calves showed (non-significant) shorter gestations, easier calvings and lower percentages of perinatal mortality and congenital malformations than IVP co-culture calves, birth weights were not decreased. In Study 2b however, the difference between IVP co-culture and IVP SOF calves in birth weight and ease of calving was significant (P < 0.05), IVP SOF calves resembling MOET calves more. IVP calves differed significantly from MOET calves with respect to several physiological parameters, such as blood oxygen saturation level, heart beat frequency and some measures of the heart. In addition, in Study 2b, recipients receiving an IVP SOF embryo showed a more regular return to estrus than those receiving an IVP co-culture embryo. From Study 2 it can be concluded that using a semi-defined medium for in vitro culture (SOF) may improve characteristics of IVP calves born.

The relationship between the number of spermatozoa inseminated and the reproductive efficiency of individual dairy bulls.

The semen of 20 mature, evaluated bulls was split-sample diluted and contained 2.1 x 10(6) to 17.3 x 10(6) total spermatozoa per 0.25-ml French straw. The number of viable inseminated spermatozoa ranged from 1.1 x 10(6) to 11.8 x 10(6). Each bull had 2430 to 5330 first or second inseminations performed. The nonreturn rate at 56 d after AI was estimated for every dilution. The daily nonreturn rates to 180 d were used to estimate conception and calving rates at a given concentration. The relationship was determined between these estimations and the number of spermatozoa that were actually inseminated. The bulls differed significantly in their maximal nonreturn rate at high sperm numbers per AI and in the rate at which they approached this maximum. There was no correlation between the maximum nonreturn at high sperm numbers and the rate of approach, which implies that the ranking of the bulls for nonreturn rate 56 d after AI changes with the number of spermatozoa inseminated. Multiphasic analysis of reproductive efficiency revealed bull differences in estimated conception and calving rates. The estimated calving rate after conception was 82 to 90% and was independent of the number of spermatozoa that were inseminated. The sperm numbers needed to obtain 95% of the maximal conception rate ranged from 1 x 10(6) to 11 x 10(6).

Abnormal offspring following in vitro production of bovine preimplantation embryos: a field study.

Data on 944 calves from 2228 in vitro-produced (IVP) bovine preimplantation embryos were compared with data on 2787 AI calves born in the same herds in 1995. Bovine preimplantation embryos were produced in vitro following ovum pick up (OPU) from donor cows and pregnant heifers in an open nucleus breeding program. After 7 d of in vitro culture on a BRL cell monolayer in the presence of 10% FCS, frozen-thawed expanded blastocysts and fresh morulae to expanded blastocysts were transferred into recipient heifers and cows at 119 contracted farms throughout the Netherlands. The pregnancy rate, as confirmed by palpation per rectum between 90 and 150 d after transfer was 43.5% for both fresh and frozen embryos. Data on IVP and AI calves were registered by the farmers. The percentage of calves with a congenital malformation and the percentage of male calves were related to the total number of calves born. Gestation length, birth weight (measured by a balance), perinatal mortality and ease of calving were analyzed in a subdataset (699 IVP and 2543 AI calves, respectively) by a comparative analysis of variance (ANOVA). The ANOVA model included herd, month of calving, sire nested within AI or IVP, parity and breed of the inseminated cow/embryo recipient, sex of calf, type of calf (AI or IVP) and two-way interactions between type of calf and sex, parity and breed. The percentage of calves with congenital malformations was 3.2% and 0.7% for IVP and AI calves, respectively. An increased incidence of hydro-allantois and abnormal spinal cords and limbs was observed in IVP calves. The percentage of male calves was significantly different between IVP and AI, 55.5% and 48.9%, respectively (Chi-square, 1 degree of freedom, P < 0.05). On the average, IVP calves showed a significant increase of birth weight by 10% (4-5 kg), a 3-d longer gestation period, 2.4% more perinatal mortality and a more difficult calving process compared to AI calves (P < 0.05). From these results it is concluded that calves produced by IVP deviate significantly from calves produced by AI.

Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution.

We designed and conducted a field trial to obtain accurate pregnancy rates of Day 7 bovine embryos after vitrification in PB1 containing 6.5 M glycerol and 6% BSA (w/v) and one-step dilution in 1 M sucrose compared with controlled slow freezing in 1.5 M glycerol and three-step dilution. Embryos were collected from superovulated donor cows, and Grade 1 and 2 morulae and blastocysts were randomly assigned to each cryopreservation treatment group. Dutch farmers were solicited to participate in the field trial by an advertisement that offered cryopreserved embryos at subsidized cost. Within a period of 11 wk, one of six technicians visited 150 farms. Standard nonsurgical methods were used to transfer a total of 728 cryopreserved embryos. Recipient cows, mainly multiparous and of various breeds, the so-called "bottom-end" of the national herd, received embryos either 6, 7 or 8 d after standing estrus during natural estrous cycles. We compiled a database on 22 factors that may influence establishment of pregnancy in order to check randomization of each factor over cryopreservation treatment groups and embryo transfer technicians and to perform the statistical tests. Overall pregnancy rates were 44.5% (n = 393) for vitrified embryos and 45.1% (n = 335) for slowly frozen embryos. Pregnancy rates were not significantly different (ANOVA, P = 0.79 or Chi- square analysis, P = 0.88). The registered data confirm that all factors were randomly distributed over cryopreservation methods and technicians. Technician was not a significant source of variation in pregnancy rate (analysis of variance, P = 0.79). Although three technicians performed better with the one-step procedure and the other three performed better using the three-step method, the interaction between the technician and cryopreservation method was not significant (Tukey's test for nonadditivity, P = 0.13). Our results indicate that 1) vitrification and one-step dilution can be successfully used in the field without significant reduction in the pregnancy rate and 2) the pregnancy rate obtained using the "bottom-end" of the herd is satisfactory for practical application.

Multiphasic analysis of reproductive efficiency of dairy bulls.

Reproductive efficiency of dairy bulls is usually measured by nonreturn rate. Nonreturn is a compound trait that is a result of two events, conception and gestation, that lead to calving. Nonreturn rates can be used to derive more elementary biological measures for reproductive efficiency, such as conception rate and calving rate, which separately might be more reliable than nonreturn rate itself to evaluate the fertility of a bull or the performance of an AI technician. The challenge of this study was to examine the decline in nonreturn rate in light of the theory of multiphasic analysis. A multiphasic logistic function was developed to model decline in nonreturn rate by estimating conception rate, calving rate, and characteristics of the first two estrous cycles. The model is illustrated with data on daily nonreturn rates to 120 d. From the proportion of cows that conceived but failed to complete gestation because postsignal embryonic death, the model estimates conception rate and calving rate. From the proportion of cows that failed to conceive or that conceived but failed to complete gestation because of presignal embryonic death, the model estimates the proportion of returns, or probability of detecting estrus, duration of nonreturns, and time of maximum decline in nonreturn rate for the first two cycles. Using the proposed model, conception rate and calving rate estimated from daily nonreturn rates might be more reliable for evaluation of performance of an AI technician and fertility of a bull than nonreturn rates at arbitrarily chosen days after insemination.

A model for reproductive efficiency of dairy bulls.

Reproductive efficiency of bulls is usually measured by nonreturn rate, which is commonly defined as the proportion of cows that were inseminated and did not return for another service within a specified number of days. The AI organizations use nonreturn rate to evaluate fertility of a bull or performance of a technician. Measures derived from nonreturn rate, such as conception rate and calving rate, might be more reliable for evaluation than nonreturn rate itself. Estimated conception rate is a better early measure of efficiency than nonreturn rate, because conception rate depends on the population of spermatozoa at insemination and not on developmental potential of the conceptus after insemination. A mathematical function is presented to model reproductive efficiency of bulls by estimation of the probability of conception at time of insemination (conception rate) and the probability of completing gestation after insemination (calving rate) through the relationship of nonreturn rate to the concentration of spermatozoa at insemination and the time after insemination. The model is illustrated with three bulls, using nonreturn rates by 28, 56, and 84 d after insemination.

Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos.

Day 7 bovine morulae and early blastocysts were randomly assigned to one of four cryopreservation methods: (i) a modified conventional controlled slow freezing and stepwise dilution after thawing; and three methods which enable direct transfer of the embryo into the recipient upon thawing: (ii) conventional controlled slow freezing and a modification of a one-step procedure, (iii) vitrification with 6.5 M glycerol plus 6% BSA (w/v), and (iv) vitrification with 25% glycerol (v/v) and 25% propanediol (v/v). In a comparative in vitro study, the percentage of grade 1 and 2 embryos developing into expanded blastocysts in culture for cryopreservation methods 1-4 were, respectively, 53% (29/55), 33% (20/61), 44% (26/59), and 51% (17/33). Method 2 yielded a significantly lower survival rate than methods 1 (P < 0.1) and 4 (P < 0.05) and was excluded from a subsequent test of in vivo development. Pregnancy rates (Day 60) after transfer of embryos cryopreserved by methods 1, 3, and 4 were, respectively, 59% (20/34), 43% (17/40), and 24% (5/21). Method 4 yielded a significantly lower pregnancy rate than method 1 (P < 0.05). Method 3, however, did not yield a statistically different pregnancy rate (P > 0.1) when compared to method 1. Method 3 has considerable promise in providing a successful method for the cryopreservation of bovine embryos that (i) reduces the time required for equilibration and cooling, (ii) provides for simple and rapid one-step dilution of cryoprotectant after thawing, and (iii) enables more embryos to be thawed and transferred per unit time.

Linkage analysis by two-dimensional DNA typing.

In two-dimensional (2-D) DNA typing, genomic DNA fragments are separated, first according to size by electrophoresis in a neutral polyacrylamide gel and second according to sequence by denaturing gradient gel electrophoresis, followed by hybridization analysis using micro- and minisatellite core probes. The 2-D DNA typing method generates a large amount of information on polymorphic loci per gel. Here we demonstrate the potential usefulness of 2-D DNA typing in an empirical linkage study on the red factor in cattle, and we show an example of the 2-D DNA typing analysis of a human pedigree. The power efficiency of 2-D DNA typing in general is compared with that of single-locus typing by simulation. The results indicate that, although 2-D DNA typing is very efficient in generating data on polymorphic loci, its power to detect linkage is lower than single-locus typing, because it is not obvious whether a spot represents the presence of one or two alleles. It is possible to compensate for this lower informativeness by increasing the sample size. Genome scanning by 2-D DNA typing has the potential to be more efficient than current genotyping methods in scoring polymorphic loci. Hence, it could become a method of choice in mapping genetic traits in humans and animals.

DNA profiling of cattle using micro- and minisatellite core probes.

We have evaluated 15 different micro- and minisatellite core probes for use in identity and paternity testing in cattle, based on Southern blot hybridization analysis. The core probes were tested in animals of different breeds and by analysis of seven two-generation pedigrees. Of the 15 core probes tested, seven were able to detect on average seven variant bands per individual animal. Segregation analysis showed that on average two out of 36 variant bands scored per core probe were genetically linked while two out of 12 variant bands correspond to the same allelic pair. The results obtained demonstrate the effectiveness of multilocus core probes for determining identity and paternity in cattle.

Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling and freezing.

In this study attempts were made to improve the survival rates of bull spermatozoa after freezing/thawing and to clarify the importance of certain agents to the cryopreservation of spermatozoa. For that purpose the standard freezing extender was modified by the addition of different concentrations of various cryoprotectants and membrane-stabilizing agents: glycerol, 1,2-propanediol, polyvinylpyrrolidone, sucrose, egg yolk, lipid vesicles, and bovine serum albumin (BSA). Sperm membrane impermeability toward H33258 was employed as the parameter for sperm integrity during cooling and after freezing/thawing. Exclusion of glycerol from the extender did not significantly affect sperm integrity. Replacing 6% glycerol by 6% 1,2-propanediol resulted in reduced sperm survival, whereas replacement of glycerol by 62.5 mM sucrose slightly improved survival rates. Addition of 5 or 10% polyvinylpyrrolidone (either or not in combination with 0.5 M sucrose) significantly reduced sperm integrity. Excluding egg yolk from the extender caused a serious decrease of sperm survival after both cooling and freezing. The cryoprotection offered by egg yolk could not be mimicked by dioleoylphosphatidylcholine (DOPC) vesicles or DOPC/phosphatidic acid/cholesterol vesicles in concentrations up to 29 or 9 mM, respectively. However, the freezing extender containing 6.5 mM DOPC vesicles in combination with 6% BSA yielded results which did not significantly differ from those obtained with the standard extender; higher vesicle concentrations combined with BSA might produce even better results. Further research on the cryopreservation of bovine spermatozoa should focus on membrane stabilization since the membrane-stabilizing compounds yield more promising results than the ice-preventing agents.

The fix vital stain method. Simultaneous determination of viability and acrosomal status of bovine spermatozoa.

A rapid, accurate and precise method for simultaneous determination of acrosomal status and viability of bull spermatozoa is introduced and evaluated. The method involves fixation of semen with glutaraldehyde and subsequent addition of the fluorescent dye Hoechst bisbenzimide 33258 (H33258). Wet mounts were examined using a combination of phase-contrast and fluorescence microscopy (x500) for simultaneous visualization of the acrosomal apical ridge, which is indicative of the presence of an intact acrosome, and H33258-labeled nuclei, which is indicative of membrane-damaged cells. This fix-vital stain method allows differentiation between true acrosome reactions and degenerative postmortem loss of acrosomal membranes. Incubation of frozen-thawed spermatozoa for 60 minutes at 37 degrees C in the presence of calcium ionophore A23187 resulted in an increase in the percentage of true acrosome-reacted spermatozoa. The fix-vital stain method does not contain any processing steps that result in loss, selection, or damage of spermatozoa and therefore allows evaluation of representative semen samples.