AI Bulls Research Articles

Genetic parameters for fertility of dairy heifers and cows at different parities and relationships with production traits in first lactation

The objectives of this study were to estimate genetic parameters for fertility of Brown Swiss cattle, considering reproductive measures in different parities as different traits, and to estimate relationships between production traits of first lactation and fertility of heifers and first-parity and second-parity cows. Reproductive indicators were interval from parturition to first service, interval from first service to conception, interval from parturition to conception, number of inseminations to conception, conception rate at first service, and nonreturn rate at 56 d after first service. Production traits were peak milk yield (pMY), lactation milk yield, and lactation length (LL). Data included 37,546 records on heifers, and 24,098 and 15,653 records on first- and second-parity cows, respectively. Cows were reared in 2,035 herds, calved from 1999 to 2007, and were progeny of 527 AI bulls. Gibbs sampling was implemented to obtain (co)variance components using both univariate and bivariate threshold and censored linear sire models. Estimates of heritability for reproductive traits in heifers (0.016 to 0.026) were lower than those in first-parity (0.017 to 0.142) and second-parity (0.026 to 0.115) cows. Genetic correlations for fertility in first- and second-parity cows were very high (>0.920), whereas those between heifers and lactating cows were moderate (0.348 to 0.709). The latter result indicates that fertility in heifers is a different trait than fertility in lactating cows, and hence it cannot be used as robust indicator of cow fertility. Heifer fertility was not related to production traits in first lactation (genetic correlations between −0.215 and 0.251). Peak milk yield exerted a moderate and unfavorable effect on the interval from parturition to first service (genetic correlations of 0.414 and 0.353 after first and second calving, respectively), and a low and unfavorable effect on other fertility traits (genetic correlations between −0.281 and 0.295). Infertility after first calving caused a strong elongation of the lactation, and LL was negatively correlated with fertility of cows after second calving, so that LL can itself be regarded as a measure of fertility. Lactation milk yield depends on both pMY and LL, and, as such, is a cause and consequence of (in)fertility.

Effects of Some Respiratory and Glycolytic Inhibitors on Mitochondrial Functionality in Bovine Semen

Fertility potential of spermatozoa depends on maintenance of the mitochondrial membrane potential (ΔΨm) that provides energy for sperm hyperactivation immediately prior to successful fertilization. Mitochondrial structure and integrity are associated with sperm motility and reduced fertility, and measurement ofΔΨm may be suitable for determining bull semen quality. Mitochondrial membrane potential in four commercial AI bulls was assessed using JC-1 and propidium iodide in the presence of the glycolytic inhibitors 2-deoxy-D-glucose (DOG) and iodoacetamide (IAM) and the respiratory inhibitor valinomycin(VAL) to determine the maximumm ΔΨm at minimum incubation. Flow cytometry recorded ΔΨm within a unified population for all treatments that represented sperm with low and high ΔΨm respectively. Maximumm ΔΨ was seen at 40 min incubation. Mean high fluorescence intensity (MFI) (orange) was significantly greater for untreated compared to the treated sperm, at 40 and 80 min incubation, in both fresh and frozen hawed semen. In sperm treated with VAL and IAM, ΔΨm was lowered significantly, and the proportion of sperm with high: low ΔΨm ratio was higher in control and DOG- treated samples representing more active mitochondria. In samples treated with VAL and IAM, the ratio was reversed, representing loss in activity. Cryopreservation increased the high:low ΔΨm ratio only in bull 1 by 30% and lowered it in bulls 2, 3 and 4 by 20% - 70% compared to fresh semen. The rise in bull 1 may relate to be the product of sperm demonstrating a capacitation- like effect of the freeze-thaw process which stimulated sperm hyperactivity. We conclude that mitochondrial function was affected adversely on freeze-thaw process. The 40 min incubation is satisfactory for future studies. Furthermore, IAM, a known glycolytic inhibitor, has a similar effect to VAL, a recognized respiratory inhibitor, which should provide a basis for further research. © Shahani et al.

Moribund sperm in frozen-thawed semen, and sperm motion end points post-thaw and post-swim-up, are related to fertility in Holstein AI bulls

The objectives were to compare testicular physical characteristics and post-thaw sperm characteristics and their associations with fertility in Holstein bulls used for AI. Ten Holstein bulls (4–5 y old) were classified as either high-fertility (HF) or low-fertility (LF; n = 5 each), based on adjusted 56-d non-return rates [non-return rate (NRR); range (mean ± SD): 55.6 ± 4.6 to 71.8 ± 1.3%). Testicular physical characteristics were not significantly different between the two groups. Four ejaculates were collected from each bull and cryopreserved. Several indexes of sperm motion (based on computer-assisted sperm analysis) at post-thaw and post-swim-up were correlated with NRR. Sperm from HF bulls were in transition to a hyperactivated motility pattern, whereas those from LF bulls had only a forward progressive motility pattern. In HF vs LF bulls, there was a greater percentage of viable sperm after thawing (60.6 ± 9.7 vs 49.5 ± 8.0%, P < 0.05) and after swim-up (70.9 ± 11.0 vs 63.0 ± 8.8%, P < 0.01); these two end points were positively correlated with fertility (r = 0.45, P < 0.01 and r = 0.78; P < 0.01, respectively). Furthermore, in HF vs LF bulls, the ratio of sperm recovered after swim-up to viable sperm in post-thaw semen was higher (P < 0.001), and the proportion of moribund sperm expressed as a percentage of live sperm differed (12.6 ± 3.4 vs. 16.4 ± 3.1%, P < 0.001) and was negatively correlated (r = −0.33, P < 0.05) with fertility. In conclusion, fertility of Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but it was associated with differences in moribund sperm in the inseminate, as well as characteristics of sperm post-thaw and after swim-up.

Use of sexed semen and its effect on conception rate, calf sex, dystocia, and stillbirth of Holsteins in the United States

Use of sexed semen for artificial insemination of US Holstein heifers (1.3million breedings) and cows (10.8million breedings) in Dairy Herd Improvement herds was characterized by breeding year, parity, service number, region, herd size, and herd milk yield. Sexed semen was used for 1.4, 9.5, and 17.8% of all reported breedings for 2006, 2007, and 2008, respectively, for heifers, and for 0.1, 0.2, and 0.4%, respectively, for cows. For 2008 sexed semen breedings, 80.5 and 68.6% of use was for first services of heifers and cows, respectively. For cows, 63.1% of 2008 sexed semen use was for first parity. Mean sexed semen use within herd was the greatest for heifers in the Southwest (36.2%) and for cows in the Mideast (1.3%). Mean sexed semen use increased for heifers but changed little for cows as either herd size or herd mean milk yield increased. Availability of sexed semen was examined for Holstein bulls in active AI service; of 700 bulls born after 1993, 37% had sexed semen marketed by mid August 2009. Active AI bulls with marketed sexed semen were superior to average active AI bulls for evaluations of yield traits, productive life, somatic cell score, daughter pregnancy rate, service-sire calving ease, service-sire stillbirth, final score, sire conception rate, and lifetime net merit. The effect of sexed semen use on conception rate, calf sex, dystocia, and stillbirth also was examined for heifers and cows. Mean conception rate for heifers was 56% for conventional and 39% for sexed semen; corresponding conception rates for cows were 30 and 25%. For single births from sexed semen breedings, around 90% were female. Dystocia and stillbirth were more frequent for heifers (6.0 and 10.4%, respectively, for conventional semen; 4.3 and 11.3%, respectively, for sexed semen) than for cows (2.5 and 3.6%, respectively, for conventional semen; 0.9 and 2.7%, respectively, for sexed semen). Difficult births declined by 28% for heifers and 64% for cows with sexed semen use. Stillbirths were more prevalent for twin births except for sexed semen heifer breedings. Stillbirths of single male calves of heifers were more frequent for breedings with sexed semen (15.6%) than conventional semen (10.8%); a comparable difference was not observed for cows, for which stillbirth frequency of single male calves even decreased (2.6 vs. 3.6%). Overall stillbirth frequency was reduced by sexed semen use for cows but not for heifers.

Acute BRSV Infection in Young AI Bulls: Effect on Sperm Quality

Bovine respiratory syncytial virus (BRSV) infection is an important part of the calf pneumonia complex, occasionally affecting even adult cattle. However, the pathogenicity of BRSV in animals older than 6 months is often neglected. Finland is free of many contagious diseases in farm animals, and this gives a good opportunity to study the effects of specific pathogens on bovine reproduction. This report describes the deteriorating effects of BRSV epizootics on sperm morphology and fertility of young dairy bulls (n = 79) at a bull station. More than half of the young bulls had a clinical respiratory disease caused by BRSV during their quarantine when they were 6 months old. Four of seven subsequent quarantine groups were affected. Six months later, when these seropositive bulls (n = 54) came into semen production, they had poorer sperm morphology, and the proportion of normal spermatozoa was 74.1% in BRSV-seropositive animals compared with 81.2% in seronegative bulls (n = 25) (p = 0.035). Field fertility was also slightly affected, the 60-day non-return rates were 75.2% and 76.8% for BRSV seropositive and seronegative bulls respectively (p = 0.014). Potential reasons for lowered sperm quality are discussed here.

Restriction fragment length polymorphism of a bovine T-cell receptor ß gene

Genetic polymorphism of a bovine T-cell receptor beta gene was investigated by analysing restriction fragment length polymorphism (RFLP). One locus, denoted TCRB, with four allelic variants was revealed. The relationship between alleles at the TCRB locus and bull breeding values for disease and milk production was investigated in a sample of 196 progeny-tested AI bulls of the Swedish Red and White breed. The statistical evaluation of the data revealed no convincing association between TCRB alleles and any of the traits studied.

Verification for the control of kappa-casein gene on milk protein yield of Friesian-Holstein in dairy central region of West Java.

Genotipe of BB kappa kasein (ĸ-kasein) gene positively influences high milk protein yield. The aim of this research was to verify the use of the к-casein gene as marker of assisted selecion (MAS) of high protein yield in domestic Holstein-Friesian (HF) cattle. Genotyping blood samples was conducted for lactating cows from BPPT-SP Cikole (82 head) and a number of small holders in KPSBU Lembang (190 head). Genotyping the к-casein gene was also conducted for active and non active AI bulls (HF) from BIB Lembang (25 head) and BBIB Singosari (32 head). Varian genotipes were identified trough DNA migration, whereas frequencies of alleles were calculated by Nei Method (1987). The effects of variant genotypes on protein yield and other milk component yields were studied in lactating cows (56 head in BPPT-SP Cikole and 111 head in KPSBU Lembang) by applying Least Square Means (LSM) method of the General Linear Model (GLM). Genotyping the ĸ-kasein gene resulted in three genotypes, namely AA, AB and BB, with two alleles, namely A and B. It was identified that a very limited number of lactating cows with BB genotype (0-9%). These cows produced higher protein yield around 3.37-3.84% than that of AA cows, while AB cows produced protein yield in between, though these differences were not statistically significant (P>0,05). In contrast, AA cows tended to produced higher milk fat yield than BB cows. Cows BB and AB seemingly produced higher dry matter, while both specific density and pH were not affected by variant genotypes of the к-casein gene. The results showed that BB genotype of the к-casein gene tended to be quitely consistent in controlling high protein yield, so it would be a good oppurtinity to be used as MAS of milk protein yield in HF domestic. Key words : Protein Yield, Dairy Cattle (HF), к-Casein Gene, Genotype BB

26 DNA METHYLATION PATTERNS ARE APPROPRIATELY ESTABLISHED IN THE SPERM OF BULLS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER AFTER PASSAGE THROUGH THE GERMLINE

Cloning of domestic animals by somatic cell nuclear transfer (SCNT) has permitted the rescue of valuable genetics and has the potential to allow rapid dissemination of desirable traits in production animals through the use of cloned sires. Whilst cloned animals may show developmental deviations and aberrant DNA methylation suggestive of incomplete nuclear reprogramming, it is widely accepted that their offspring are normal, as any aberrant epigenetic marks are believed to be corrected on passage of the genome through the germline. We assessed the extent of reprogramming by comparing DNA methylation patterns in sperm of SCNT bulls (n = 4) with sperm from bulls generated by AI (n = 5) and with the nuclear donor somatic cells (adult skin fibroblasts). The genomic regions examined were 3 repetitive sequences (satellites 1, 2, and alpha) and CpG islands in 5 genes [HAND1, LIT1, MASH2, IGF2, Dickkopf-1(DKK-1)]. Semen was collected from 16-month-old bulls and assessed for volume, sperm number, morphology, and motility. DNA was extracted from washed sperm and somatic donor cells, bisulfite-treated and processed for quantification of CpG methylation using the Sequenom MassArray system. Methylation levels at individual CpG sites/groups of CpGs were compared between sample groups using the t-test with pooled variances. No apparent difference was detected in semen characteristics between SCNT and AI bulls. Sperm DNA methylation levels were very low in single copy genes with the exception of the CpG island in IGF2, which has previously been shown to be completely methylated in sperm. At all genomic regions examined, each CpG site or CpG groups were methylated to different levels, and each region had a distinctive profile, which was almost invariant between individual sperm samples from either the SCNT or AI bulls. In all sites examined, there were no significant differences in methylation profiles between sperm from SCNT and AI bulls. In contrast, DNA methylation profiles were significantly different between SCNT bull sperm and the donor cells. The exception was the CpG island in MASH2, which was essentially unmethylated in both. For the 3 satellite sequences along with LIT1, HAND1, and to a lesser extent, the DKK-1 region, DNA was significantly less methylated in sperm than in the donor cells. Only IGF2 was significantly more methylated in SCNT and AI sperm than in the donor cells at 10/25 CpG sites (P &lt; 0.02). The results indicate that gametes from SCNT bulls had different epigenotypes from the donor somatic cells. This is the first molecular evidence that donor cell genomes have been reprogrammed in these SCNT bulls and that after going through the germline had acquired DNA methylation profiles that were similar to AI-derived bulls. It also suggests that any epigenetic aberrations that SCNT bulls may harbor are unlikely to be passed on to their offspring through their gametes. Supported by FRST contract C10X0311.

Early oocyte penetration can predict the efficiency of bovine embryo production in vitro

The aim of this work was to characterize oocyte fertilization and embryo cleavage in nine AI bulls to find parameters suitable for prediction of in vitro fertility. According to the d8 blastocysts rate, they were categorized as high, medium and low productive (HP, MP and LP, mean: 25.4, 21.0 and 13.6% respectively) bulls. For these categories, oocyte penetration and fertilization efficiency were assessed at 6 and 18 hours post insemination (hpi), respectively. Some presumptive zygotes were cultured and cleaved and fast-cleaved embryo rates were checked at 44 hpi. The penetration rate was significantly higher for HP bulls than for MP and LP bulls (67.9 versus 50.3 and 33.1%; p<0.01). The syngamy rate was significantly higher for HP bulls than for MP and LP bulls (21.4 versus 10.2 and 5.7%; p<0.05). Conversely, no significant differences in fertilization rates were found among HP, MP and LP bulls. The cleavage rate was significantly higher for HP than LP bulls (82.4 versus 74.4%; p<0.01). The fast cleavage rate was significantly higher for both HP and MP bulls, as compared with LP bulls (82.1 and 84.7 versus 73.5%; p<0.01). A strong correlation was found between blastocyst production and penetration (r=0.803), syngamy (r=0.826), cleavage (r=0.635) and fast cleavage (r=0.709). In conclusion, all the evaluated parameters showed a predictive value, the most significant being early penetration and syngamy.

Evaluation of chromatin integrity of motile bovine spermatozoa capacitatedin vitro

The efficiency of in vitro embryo production is highly variable amongst individual sires in cattle. To eliminate that this variability is not caused by sperm chromatin damage caused by separation or capacitacion, chromatin integrity was evaluated. Seventeen of AI bulls with good NRRs but variable embryo production efficiency were used. For each bull, motile spermatozoa were separated on a Percoll gradient, resuspended in IVF-TALP medium and capacitated with or incubated without heparin for 6 h. Samples before and after separation and after 3-h and 6-h capacitacion or incubation were evaluated by the Sperm Chromatin Structure Assay (SCSA) and the proportion of sperm with intact chromatin structure was calculated. Based on changes in the non-DFI-sperm proportion, the sires were categorized as DNA-unstable (DNA-us), DNA-stable (DNA-s) and DNA-most stable (DNA-ms) bulls (n=3, n=5 and n=9, respectively). In DNA-us bulls, separation produced a significant increase of the mean non-DFI-sperm proportion (p <or= 0.01), as compared with the value before separation. Capacitacion produced a significant decrease in the mean non-DFI-sperm proportion in H+ sperm (p <or= 0.01). In DNA-s bulls, separation significantly increased the mean non-DFI-sperm proportion (p <or= 0.01) but during capacitacion, the mean non-DFI-sperm proportion remained almost unchanged. In DNA-ms bulls, neither separation nor capacitacion had any effect on the mean non-DFI-sperm proportion. It can be concluded that, although separation and capacitacion may produce some changes in sperm chromatin integrity, these are not associated with different in vitro fertility of the bulls involved.

Transmission of bovine viral diarrhoea virus through the semen of acutely infected bulls under field conditions

Epidemiological investigations implicated the semen of artificial insemination (ai) bulls as the only plausible source of infection with bovine viral diarrhoea virus (bvdv) in 10 Finnish dairy herds. The infection...

Genetic Parameters for Semen Production Traits in Austrian Dual‐Purpose Simmental Bulls

Genetic parameters were estimated for semen production traits collected in an Austrian AI centre in the years 2000-2004. In total, 12,746 ejaculates from 301 Austrian dual-purpose Simmental (Fleckvieh) AI bulls were examined considering different effects on ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. The model for genetic parameter estimation included the fixed effects age of bull, collection interval, number of collections on collection day, bull handler, semen collector, year and month of collection, a random additive genetic component and a permanent environmental effect. Correlations between estimated breeding values for semen traits and male fertility from the routine evaluation were calculated. The fertility trait considered in the routine evaluation is non-return rate 90 for the first insemination. All semen production traits were moderately heritable. Heritabilities for volume, concentration, percentage of viable spermatozoa, total number of spermatozoa and motility were 0.18, 0.14, 0.10, 0.22 and 0.04, respectively. Correlations between breeding values for semen quality traits and routinely estimated breeding values for male fertility were low and ranged from 0.08 to 0.17 indicating that semen production traits are rather poor predictors of male fertility.

Epidemiology and eradication of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV) virus in Finland

BackgroundInfectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV) is a significant disease among domestic and wild cattle. The BHV-1 infection was first detected in Finland in 1970; presumably it was imported in 1968. The infection reappeared in the large-scale bulk-tank milk surveillances which started in 1990, and was eradicated in 1994. Our aim is to describe the epidemiology of this infection in Finland, and its eradication.Materials and methodsThe official sources of pertinent information, the legal basis for the disease control and the serological methods for the detection of the infection are described.Results and conclusionTen AI bulls were found to be seropositive in 1970–1971. The total number of herds with BHV-1 antibody positive animals in the large-scale surveillance in 1990 and subsequent epidemiological investigations in 1991 was five, and the total number of seropositive animals was 90. The five herds formed three epidemiological units; semen of at least one bull seropositive in 1971 had been used in each unit. This remained the only plausible route of infection in each of the three units. Using the 'test and slaughter' approach and total stamping out in one herd the infection was eradicated in 1994.

Changes in Semen Quality in Estonian Holstein AI Bulls at 3, 5 and 7 years of Age

The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.

Effect of post-ovulatory PGF2alphaalpha or cloprostenol on plasma progesterone concentrations in mares

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1–11 before collection) and during spermatogenesis (average temperature of days 12–65 before collection). Age of bull significantly affected all traits (P < 0.01 to P < 0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly (P < 0.05 to P < 0.001) while collection intervals between 4–9 days and 1–6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively (P < 0.10 to P < 0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate (P < 0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5–15 °C were found to be optimal for semen production.

288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P &amp;lt; 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 � 9.5% vs. 21.9 � 6.7%; ANOVA: P &amp;lt; 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 � 2.1 hpi vs. 25.4 � 2.7 hpi for bull B; ANOVA: P &amp;lt; 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P &amp;lt; 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P &amp;lt; 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.

Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1–11 before collection) and during spermatogenesis (average temperature of days 12–65 before collection). Age of bull significantly affected all traits ( P < 0.01 to P < 0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly ( P < 0.05 to P < 0.001) while collection intervals between 4–9 days and 1–6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively ( P < 0.10 to P < 0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate ( P < 0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5–15 °C were found to be optimal for semen production.

113 THE DISTRIBUTION OF THE LEPTIN PROTEIN WITHIN BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS MATURED AND FERTILIZED IN VITRO

It has recently been documented that leptin regulates processes linked to reproduction including preimplantation development, embryo implantation (trophoblast invasion), and fetal growth. Transcripts for the leptin gene (LEP) and the leptin receptor gene (LEPR) have been identified in ovary, testis, placenta, endometrium, ovarian follicles, and oocytes, and also in mouse, rat, human, and bovine pre-implantation embryos. Moreover, the leptin protein was detected in mouse and human oocytes and embryos, and its localization was polarized. The distribution of regulatory proteins within oocytes and pre-implantation embryos is critical for early mammalian development, such as determination of the animal pole and the establishment of the trophoblast and the inner cell mass cells (ICM). So far there is no published evidence concerning this phenomenon in bovine oocytes and embryos. Therefore, the aim of this work was to analyze the leptin protein distribution within bovine oocytes and preimplantation embryos matured and fertilized (in vitro). The material for this work consisted of oocytes collected from slaughterhouse ovaries and sperm collected from AI bulls. In vitro oocyte maturation and fertilization were carried out according to the method described by Makarevich and Markkula (2002 Biol. Reprod. 66, 386–392). The preliminary experiment of leptin protein localization by immunofluorescent staining included immature and matured oocytes and blastocysts. Oocytes and embryos were fixed in PBS containing 4% paraformaldehyde and reacted with affinity-purified polyclonal rabbit primary antibody directed against leptin (0.1 mg/mL; Ob (Y20), Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); and then exposed to secondary goat-anti-rabbit antibody (1.0 mg/mL; Santa Cruz Biotechnology Inc.)-fluorescein isothiocyanate (FITC) conjugate. Finally, chromatin was visualized by propidium iodide staining (0.5 μg/mL). Slides were examined under a conventional fluorescence microscope (Nikon) and confocal microscope (Zeiss). The preliminary results demonstrate that the distribution of leptin differed between immature and mature oocytes: it was spherical in immature oocytes (a rim beneath the oolemma) whereas it became evenly distributed after maturation. In blastocysts, leptin signals were present inboth the trophoblast cells and in the ICM cells. This is in contrast with studies on mouse embryos which showed the presence of the LEP protein in the trophoblast only. Future experiments will include studies of embryos at the 2-cell, 4-cell, 8–16-cell, and morula stages. The present study for the first time shows the pattern of leptin protein distribution within bovine oocytes and preattachment embryos.

238 ASSESSMENT OF SPERM MOTILITY, VIABILITY, AND FERTILIZATION RATE OF TWO GROUPS OF BULLS SHOWING A DIFFERENT ABILITY TO PROMOTE EMBRYO DEVELOPMENT IN VITRO

Low developmental rates after IVF with some AI bulls are one of the most frequent problems encountered with in vitro production of bovine embryos. In this study, two groups of high and low in vitro fertility bulls (3 bulls per group) were selected based upon blastocyst rates. We first evaluated sperm motion characteristics using computer-assisted sperm analysis (CASA) before and after Percoll separation. The following parameters were compared between bulls: proportion of motile sperm (% MOT), curvilinear velocity (VCL), and straightness (STR), which allowed us to evaluate the proportion of progressive semen (VCL &gt; 80 μm/s and STR &gt; 60%). Concerning % MOT, differences between bulls were observed before Percoll separation (42 to 59% – ANOVA2: P = 0.017). Following separation, the values of % MOT were globally increased (ANOVA3: P &lt; 0.001) and no further difference was observed between bulls (68 to 76%). The same pattern was obtained for the proportion of progressive spermatozoa (before: 43 to 55% vs. after: 48 to 57% – ANOVA3: P = 0.005). For viability measurements, two DNA-binding fluorochromes, propidium iodide and Hoechst 33342, were used, which respectively stain dead sperm nuclei in red and living sperm nuclei in blue. This viability evaluation was carried out after 0, 2, 6, and 18 h of incubation in TALP medium supplemented with heparin (10 μg/mL), under the same conditions as IVF but without oocytes. In the two groups of bulls, a sharp decrease in the proportion of living sperm was already observed at 2 h (only 50 to 20% of living sperm), while, at 18 h, 17 to 9% of sperm were still intact. Two bulls from the high in vitro fertility group showed a percentage of living sperm significantly more important at 6 and 18 h than the other 4 bulls (chi square – P &lt; 0.05). In a third experiment, we assessed the fertilization (2 pronuclei) and the polyspermy (&gt;2 pronuclei) rates after staining the zygotes with Hoechst at 18 hpi. The high fertility group showed higher fertilization rates than the low fertility one (75 to 80% vs. 59 to 67% – ANOVA2: P &lt; 0.008). No significant difference was noticed between bulls concerning polyspermy. The present results confirm the efficiency of Percoll separation in enhancing some of the motility parameters such as the proportion of motile and progressive sperm. Moreover, this gradient allows us to abolish differences between bulls for those motility parameters. However, the viability and the rate of fertilization still varied between bulls after Percoll separation. In our study, only fertilization rates seemed related to the blastocyst rates. Further investigations are under way to better understand the sperm characteristics of these two groups. This work was supported by the Minestery of Agriculture of the Region wallonne de Belgique.

Short Communication: Effect of Leptin Gene Polymorphisms on Breeding Value for Milk Production Traits

New molecular techniques focused on genome analysis open new possibilities for complex evaluation of economically important traits in farm animals. Milk production traits are typical quantitative characteristics controlled by a number of genes. Mutations in their sequences may alter animal performance as well as their breeding values. In this study, we investigated the effect of 3 restriction fragment length polymorphisms (RFLP): HphI, Kpn2I, and Sau3AI in the leptin gene, on bull breeding values for milk, fat, and protein yield, and fat and protein content. One hundred seventeen Polish Black and White AI bulls were genotyped. Pedigree analysis indicated a relatively close relationship between the bulls. Statistical analysis indicated that the HphI polymorphism has a significant effect on milk and protein yield. Animals with the TT genotype had approximately 2× higher estimated breeding values for milk and protein yields. No effect was found for the other 2 polymorphisms.