Spermatozoa progressive motility is positively correlated with fertilization competence. Bulls’ ejaculates with progressive motility lower than 50% are routinely rejected through the process of straw preparation, designated for artificial insemination of dairy cows. We examined the quality and fertility competence of ejaculates with relative low progressive motility (55–60%, n = 5; control) with those of very low progressive motility i.e. below the lower threshold, (20-45%, n = 5; rejected). Analysis revealed a lower volume for the control vs. rejected samples. Dip-Quick staining revealed a higher proportion of spermatozoa with abnormal morphology in the rejected group, in particular those with detached heads. Activation of spermatozoa with calcium ionophore, resulted by a lower proportion of activated spermatozoa in the rejected group. In addition, a higher proportion of spermatozoa with DNA damage were recorded in the rejected vs. the control samples. Following in-vitro fertilization, the proportion of oocytes that developed to the 2- and 4-cell stage embryos did not differ between groups. However, the proportion of embryos that further developed to blastocysts, was higher in the control group. Transcript abundance of selected genes in the blastocysts and the apoptotic index did not differ between groups, suggesting that the forming blastocysts were of the same quality. It is suggested that in specific cases, for example genetically superior bulls, ejaculates with very low progressive motility can be used for in vitro production of embryo. Further in vivo examinations, i.e. artificial insemination or transferring of embryos derived from these inferior ejaculates, might clarified this point.

Intensive reproductive management in dairy herds is mostly based on AI using high-merit bulls. Therefore, semen quality of bulls is of high importance. An association between semen quality and fatty acid content in feed has been suggested. Accordingly, the aim of this study was to examine the effect of omega-3 supplementation on sperm traits and fertilization competence. Fifteen Israeli Holstein bulls were assigned to three experimental groups. Bulls were fed over 13 weeks with a standard ration top-dressed with encapsulated-fat supplementation-fish oil (FO) or flaxseed oil (FLX; i.e. omega-3 sources), or saturated fatty acids (SFA, control). Ejaculates were collected before initiation of the study, during the feeding trial, and 1 month after feeding trial. Ejaculates were treated according to the routine procedure of the Israeli AI centre (Sion Ltd.), frozen, and stored in straws. Frozen-thawed samples were subjected to “swim-up” procedure, and spermatozoon viability, mitochondrial membrane potential, reactive oxygen species (ROS) level, acrosome membrane integrity, and DNA fragmentation were evaluated via flow cytometry, using sperm-specific kits (EasyCyte, IMV Technologies). Feeding with FO, FLX, or SFA did not affect the viability or mitochondrial membrane potential of sperm collected before, during, or after the feeding trial. On the other hand, a reduced proportion of sperm with ROS expression was recorded in the FLX samples compared to the SFA sample at the end of the feeding trial (42.2±1.2 vs. 47.3±4.3%, respectively; P<0.05) and one month later (36.3±2.2 vs. 41.6±4.6%, respectively; P<0.05). A low proportion of sperm with damaged acrosomal membrane was observed in both FLX and FO samples compared with SFA at the end of the feeding trial (48.8±3.4 and 41.7±2.7 vs. 59.8±3.4%, respectively; P<0.05). The proportion of sperm with fragmented DNA was lower in the FLX group than in the SFA group, collected one month after the end of the feeding trial (2.3±0.6 vs. 5.4±1.2%, respectively; P<0.05). To examine fertilization competence, oocytes were aspirated from ovaries collected from a local abattoir, then matured (n=216; 3 replicates) and fertilized invitro with a pool of samples from each group, collected one month after the end of the feeding trial (n=5 samples per group). The proportions of 2- to 4-cell-stage embryos and of blastocysts were determined 42h and 8 days after fertilization, respectively. Although the proportion of cleaved embryos did not differ between groups, a higher blastocyst formation rate was recorded in the FLX group (P<0.05), and a moderate increase was noted in the FO group, relative to the SFA group (28.1±4.4, 19.1±2.6, and 11.9±3.4%, respectively). Results imply that feeding bulls with omega-3 originating from FLX improves sperm quality, most likely due to improved redox status and decreased DNA fragmentation. This nutritional approach seems to be an effective tool for improving bull fertility competence. Nevertheless, invivo examination is required.

SummaryWe examined the association between progressive motility of spermatozoa andin vitrofertilization (IVF) competence of bovine ejaculates. Fresh semen was evaluated using a computerized sperm quality analyzer for bulls using progressive motility as the primary parameter. Ejaculates with high progressive motility (HPM; >81%) were compared with those with low progressive motility (LPM; <62%). Semen concentration and sperm velocity were lower (P< 0.05) in HPM versus LPM ejaculates. Volume and motile sperm concentration did not differ between groups (P> 0.05). Examination of sperm morphology revealed a higher proportion of spermatozoa with abnormal morphology (P< 0.01) in LPM versus HPM ejaculates, the predominant abnormal feature being a bent tail (P< 0.05). Sperm viability, acrosome integrity and DNA fragmentation did not differ between HPM and LPM samples. Mitochondrial membrane potential was higher (P< 0.01) in HPM versus LPM semen. Zinc concentrations in the seminal plasma correlated with progressive motility (R2= 0.463,P= 0.03). In addition, representative ejaculates from HPM and LPM groups were cryopreserved in straws and used for IVF. The proportions of embryos cleaved to 2- and 4-cell stages (88.1 ± 1.1 versus 80.5 ± 1.7,P= 0.001) and developed to blastocysts (33.5 ± 1.6 versus 23.5 ± 2.2,P= 0.026) were higher for HPM than LPM semen. The total cell number of embryos and blastocyst apoptotic index did not differ between groups. Although sperm progressive motility is associated with IVF competence, further examination is required to determine whether progressive motility can serve as a predictor of semen fertilization capacityin vivo.

Several studies have shown that computation of genomic estimated breeding values (GEBV) with accuracies significantly greater than parent average (PA) estimated breeding values (EBVs) requires genotyping of at least several thousand progeny-tested bulls. For all published analyses, GEBV computed from the selected samples of markers have lower or equal accuracy than GEBV derived on the basis of all valid single nucleotide polymorphisms (SNPs). In the current study, we report on four new methods for selection of markers. Milk, fat, protein, somatic cell score, fertility, persistency, herd life and the Israeli selection index were analyzed. The 972 Israeli Holstein bulls genotyped with EBV for milk production traits computed from daughter records in 2012 were assigned into a training set of 844 bulls with progeny test EBV in 2008, and a validation set of 128 young bulls. Numbers of bulls in the two sets varied slightly among the nonproduction traits. In EFF12, SNPs were first selected for each trait based on the effects of each marker on the bulls' 2012 EBV corrected for effective relationships, as determined by the SNP matrix. EFF08 was the same as EFF12, except that the SNPs were selected on the basis of the 2008 EBV. In DIFmax, the SNPs with the greatest differences in allelic frequency between the bulls in the training and validation sets were selected, whereas in DIFmin the SNPs with the smallest differences were selected. For all methods, the numbers of SNPs retained varied over the range of 300 to 6000. For each trait, except fertility, an optimum number of markers between 800 and 5000 was obtained for EFF12, based on the correlation between the GEBV and current EBV of the validation bulls. For all traits, the difference between the correlation of GEBV and current EBV and the correlation of the PA and current EBV was >0.25. EFF08 was inferior to EFF12, and was generally no better than PA EBV. DIFmax always outperformed DIFmin and generally outperformed EFF08 and PA. Furthermore, GEBV based on DIFmax were generally less biased than PA. It is likely that other methods of SNP selection could improve upon these results.

Season-induced variation in fatty acid and cholesterol composition in bovine semen has been associated with semen quality. Given the specific roles of the various semen compartments (seminal fluids, sperm head, and sperm tail) in fertilization, we hypothesized that environmental-stress-induced alterations in the lipid composition of a specific compartment might impair semen quality and sperm function. Semen samples were collected from five mature Holstein-Friesian bulls during the summer (August to September) and winter (December to January). Semen was evaluated by computerized sperm-quality analyzer, calibrated for bulls' semen, and centrifuged to separate the spermatozoa from the seminal fluids. The spermatozoal fraction was sonicated to separate the sperm head and tail compartments. Cold lipid extraction was performed with chloroform:methanol (2:1, vol/vol). Lipids were identified and quantified by gas chromatography. Seasonal variation was found in both physiological and structural parameters. The proportion of spermatozoa defined as morphologically normal was higher in the winter, with higher motility, progressive motility, and velocity relative to summer samples. Lipid composition within fractions varied between seasons with prominent impairment in the tail compartment, characterized by high saturated fatty acid, low polyunsaturated fatty acid, and low cholesterol concentrations during the summer. Given the association between alterations in lipid composition and reduced sperm motility and velocity during the summer, it is suggested that lipid composition might serve to predict sperm quality.

Based on pairwise identity-by-state (IBS) distances and whole-genome SNP data, kinship was investigated in the Israeli Holstein population. A total of 789 bulls, including most of the artificial insemination sires in service since 1987, were genotyped by the BovineSNP50 BeadChip. This sample included up to five generations. For each bull-by-bull combination, three states are possible for each marker: no match, a single match and both alleles match. Summing over all markers, the 932 598 IBS scores (three match frequencies*310 866 bull-by-bull combinations) were visualized using three-dimensional coordinates that corresponded to the frequencies of the three possible states. Results were reduced to two dimensions using the transformations x' = 0.7071(1 + freq1-freq2) and y' = 1.2247freq0. Bull-by-bull pairs were grouped according to their level of kinship, and canonical scores were calculated using discriminant analysis and the x' and y' features. Of the 474 pairs of recorded maternal grandsire-grandson with both individuals genotyped, the probability for 28 pairs to belong to this level of kinship was low (P < 0.05), suggesting an error rate of around 3% per generation in pedigree determination.

An efficient algorithm for genomic selection of moderately sized populations based on single nucleotide polymorphism chip technology is described. A total of 995 Israeli Holstein bulls with genetic evaluations based on daughter records were genotyped for either the BovineSNP50 BeadChip or the BovineSNP50 v2 BeadChip. Milk, fat, protein, somatic cell score, female fertility, milk production persistency and herd-life were analyzed. The 400 markers with the greatest effects on each trait were first selected based on individual analysis of each marker with the genetic evaluations of the bulls as the dependent variable. The effects of all 400 markers were estimated jointly using a 'cow model,' estimated from the data truncated to exclude lactations with freshening dates after September 2006. Genotype probabilities for each locus were computed for all animals with missing genotypes. In Method I, genetic evaluations were computed by analysis of the truncated data set with the sum of the marker effects subtracted from each record. Genomic estimated breeding values for the young bulls with genotypes, but without daughter records, were then computed as their parent averages combined with the sum of each animal's marker effects. Method II genomic breeding values were computed based on regressions of estimated breeding values of bulls with daughter record on their parent averages, sum of marker effects and birth year. Method II correlations of the current breeding values of young bulls without daughter records in the truncated data set were higher than the correlations of the current breeding values with the parent averages for fat and protein production, persistency and herd-life. Bias of evaluations, estimated as a difference between the mean of current breeding values of the young bulls and their genomic evaluations, was reduced for milk production traits, persistency and herd-life. Bias for milk production traits was slightly negative, as opposed to the positive bias of parent averages. Correlations of Method II with the means of daughter records adjusted for fixed effects were higher than parent averages for fat, protein, fertility, persistency and herd-life. Reducing the number of markers included in the analysis from 400 to 300 did not reduce correlations of genomic breeding values for protein with current breeding values, but did slightly reduce correlations with means of daughter records. Method II has the advantages as compared with the method of VanRaden in that genotypes of cows can be readily incorporated into the Method II analysis, and it is more effective for moderately sized populations.

Strong selection in the Israeli Holstein dairy cattle population over the last three decades should have left clear signatures of selection. Two experimental approaches were applied to detect evidence of contemporary selection based on the 54K BeadChip genotypes of 1000 Israeli Holstein bulls: (i) the long-range haplotype test, which searches for structural evidence resulting from selective sweep, and (ii) direct analysis of the changes in haplotypes frequencies over time combined with linkage disequilibrium blocks haplotype-based association analysis. Ten traits were analyzed: the PD07 Israeli selection index, milk, milk fat, % fat, milk protein, % protein, somatic cell score, female fertility, milk production persistency and herd life. The long-range haplotype test detected 15% of the 3288 haplotypes that showed significant positive frequency trends (P<0.05) and was significantly correlated with the substitution effects of the haplotypes and the selection intensities for the different traits. Thirty signatures of recent selection, which correspond to both approaches and affect the Israeli PD07 selection index, were identified on 17 of the 29 autosomes. The second experimental approach also was used to estimate the selection intensity of the different traits. The correlation between the selection intensities for the traits analyzed, derived from changes in haplotype frequencies in the population of bulls, and those derived from trait-based analysis of the cow population was 0.93 over all traits. Thus, the changes in haplotypes frequencies in the bulls population accurately estimate genetic trends in the general cow population and can be used to detect signatures of recent selection.

A single nucleotide polymorphism in the intergenic region upstream of the ZNF496 gene on Bos taurus chromosome 7 displayed significant population-wide linkage disequilibrium with milk protein percentage in the Israeli Holstein population. The frequency of the allele associated with increased protein concentration was 10%. This single nucleotide polymorphism was located in the promoter region from which a 10-exon transcript of the bovine and the ovine ZNF496 genes are transcribed. The gene architecture was similar to the mouse ortholog Zkscan17. A 5-exon murine antisense transcript was complementary to the 5' untranslated Zkscan17 region that included a sequence domain conserved between mouse and ruminants, suggesting a regulatory function. In the bovine ZNF496 chromosomal region, segregation of a quantitative trait locus (QTL) for milk protein percentage was confirmed in a daughter design sire family. Concordance was not obtained between QTL status of bulls and any of the polymorphisms in the functional elements of ZNF496. This excludes these variations as the causative polymorphism under the assumption of no epigenetic effect for this locus. However, ZNF496 variants were differentially expressed in bovine ovaries, and only the paternal variant was expressed in liver and kidney in a sheep family with polymorphic ZNF496 sequence. Thus, the search for the mutation underlying the minor QTL allele, which is a top economically favorable allele in Israeli Holstein cattle, may be complicated by the presence of an imprinting center in this QTL confidence interval.

Summary Incorrect paternity assignment in cattle can have a major effect on rates of genetic gain. Of the 576 Israeli Holstein bulls genotyped by the BovineSNP50 BeadChip, there were 204 bulls for which the father was also genotyped. The results of 38 828 valid single nucleotide polymorphisms (SNPs) were used to validate paternity, determine the genotyping error rates and determine criteria enabling deletion of defective SNPs from further analysis. Based on the criterion of >2% conflicts between the genotype of the putative sire and son, paternity was rejected for seven bulls (3.5%). The remaining bulls had fewer conflicts by one or two orders of magnitude. Excluding these seven bulls, all other discrepancies between sire and son genotypes are assumed to be caused by genotyping mistakes. The frequency of discrepancies was >0.07 for nine SNPs, and >0.025 for 81 SNPs. The overall frequency of discrepancies was reduced from 0.00017 to 0.00010 after deletion of these 81 SNPs, and the total expected fraction of genotyping errors was estimated to be 0.05%. Paternity of bulls that are genotyped for genomic selection may be verified or traced against candidate sires at virtually no additional cost.

Cryopreservation of embryos for transfer in outdoor conditions is a challenge; therefore a rapid and simple technique should be applied. Two different experiments were performed to develop a functional and efficient vitrification technique. Charolais (for Exp. 1) and Holstein (for Exp. 2) female cows were superovulated and artificially inseminated (AI). Seven days after AI, embryos were flushed from the uterus and vitrified. For vitrification, blastocyst were exposed to 10% vitrification solution (VS) for 3 min, transferred into 50% VS, and immediately thereafter into 87.5% VS (100% VS containing 38% (v/v) ethylene glycol (EG), 0.5 m trehalose, and 6% BSA in PBS). The embryos were then loaded into super open pulled straws (SOPS) (Minitub, Tiefenbach, Germany) in a minimum-volume droplet (0.5 μL). The SOPSs containing the embryos were sealed by applying a soldering device to the narrow end and by inserting an identification rod into the wide end. Samples were vitrified at a rapid cooling rate in LN Slush (VitMaster apparatus, IMT Ltd, Ness-Ziona, Israel). On the day of transfer, blastocysts were warmed by plunging the SOPS into the warming chamber of the device, which contained 70% ethanol at 37°C, for 5 s. The straws were then withdrawn from the warming chamber and the sealed end was cut off carefully. The embryos were immersed in a 200-μL drop of 0.6 m trehalose in PBS solution for 4 min and then transferred through a series of solutions containing decreasing concentrations (0.5, 0.4, 0.3, 0.2, and 0.1 m) of trehalose for 2 min each. Viability was evaluated according to the ability of the embryos to re-expand. Blastocysts with the highest morphology rank were selected for transfer and loaded into the transfer gun (Minitub). Holstein recipients were in estrus 6–8 days prior to transfer. In Exp. 1, all blastocysts (n = 15) re-expanded after the vitrification and warming processes and were transferred into the uteruses of 15 Holstein recipient cows. One recipient cow became pregnant and gave birth to a healthy calf. In Exp. 2, all blastocysts (n = 6) re-expanded after warming and were transferred, one each into the uterus of a recipient Holstein heifer. Three pregnancies are still ongoing. In summary, the results obtained demonstrate that plunging embryos in SOPSs into LN Slush in outdoor conditions offers a potential technique for embryo cryopreservation and transfers. Further field trails are required to examine the effect of recipient age (cow vs. heifer) and embryo breed (Charolais vs. Holstein) on the suggested procedure.

Fat supplementation in the diet influences reproductive performance of lactating ruminants. Changes in the fat supply alter fatty acid composition and this can affect physical properties of cell membranes. This study examined the effect of rumen bypass polyunsaturated fatty acid (PUFA) supplementation on oocyte quality, chilling sensitivity, and lipid phase transition in oocytes of the sheep. Ewes were fed a diet supplemented with calcium soaps of fish oil for 13 weeks. More follicles and oocytes were found in the ovaries of ewes supplemented with PUFA than in control ewes. The number of high-quality oocytes was higher in ewes fed PUFA than in control ewes (74.3 and 57.0%, P < 0.05, respectively). Evaluation of phospholipid fatty acid composition indicated that PUFA were present in small proportions in oocytes, and eicosapentaenoic acid and docosahexaenoic acid were absent. Supplementation with PUFA increased the proportion of long chain unsaturated fatty acid in the plasma and cumulus cells phospholipids by 7.4 and 12.7%, respectively (P < 0.05). Integrity of oocyte membranes following chilling (16degreesC, 15 min) was improved by PUFA supplementation increasing from 62.5 to 90.0% (P < 0.05). Due to changes in the oocyte's fatty acid profile, physical properties of the membrane were changed and the midpoint temperature of lipid transition reduced by 11degreesC. These results suggest that supplementation of rumen bypass PUFA to ruminant diets can change fatty acid composition of follicle components and influence parameters such as number and quality of oocytes and their chilling resistance. (C) 2002 Wiley-Liss, Inc.

We have examined the effects of combinations between egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes with either bovine spermatozoa or oocytes on cellular chilling sensitivity, lipid phase transition temperature (Tm), and the ability of the oocytes to develop to the blastocyst stage. Spermatozoa and oocytes were exposed to EPC and DPPC liposomes at various temperatures (spermatozoa: 4, 12, 16, and 25°C; oocytes: 4, 16, and 32°C). The membrane integrity of the spermatozoa-control group decreased significantly following exposure to 16 or 12°C, compared to ambient temperature (25°C). In contrast, the EPC-sperm group had a greater resistance to chilling at each temperature and showed a decline in membrane integrity only at the lowest temperatures investigated. However, the DPPC-sperm group was injured significantly at all temperatures tested. Similar to the sperm, oocytes from the control group that were exposed to 16°C were injured more severely than oocytes that were electrofused with EPC or DPPC liposomes. The membrane integrity of the oocytes at 16°C that were electrofused with either EPC or DPPC liposomes was approximately the same as the control group held at 32°C (normalized to 100%), compared to 46% in the control group at 16°C (P<0.01). The transition temperatures of the sperm and oocyte membranes revealed different Tm for the different liposome treatments. All groups had a significantly higher cleavage rate, as well as increased blastocyst formation when oocytes were exposed to temperatures above or below their Tm. We suggest that the Tm of spermatozoa or oocytes can be changed by spontaneous association or electrofusion of liposomes with cellular membranes and, consequently, the chilling sensitivity can be altered. The resulting possibility is that embryo development after cryopreservation could be improved with such a method.

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes. Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws. All freezing was done in a "Multi thermal gradient" (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) x (V). Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode. We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method. The results showed a 75 +/- 5% post thaw motility after freezing a 12 mL test tube and 50 +/- 5% after a second freezing/thawing in mini-straws, respectively. Controlled vapour freezing showed a 60 +/- 10% post thaw motility. The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 45.5% (56/123) for the controlled group. These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume.

Follicle dynamics and oocyte viability in Holstein primiparous and multiparous cows and the relationships between fertility and the biochemical and physical properties of oocyte membranes with season were examined. The conception rates of primiparous (n = 70 885) and multiparous (n = 143 490) cows differed, peaking in the winter and decreasing in the summer. The number of follicles 3-8 mm in diameter per ovary was higher in winter (19.6) compared with summer (12.0). However, in winter the percentage of ovaries with fewer than ten follicles per ovary was 16%, in contrast to 50% in summer. After aspiration of follicles, 7.5 oocytes per ovary were found in winter and 5.0 oocytes per ovary in summer. Cleavage to the two- to four-cell stage after chemical activation was greater in winter than in summer; this was enhanced at the morula stage and embryo development to the blastocyst stage was significantly higher in winter than in summer. Determination of the lipid phase transition in oocyte membranes revealed a shift of 6 degrees C between summer and winter. Fatty acid composition of phospholipids from follicular fluid, granulosa cells and oocytes indicated that there was a higher percentage of saturated fatty acids during the summer and that the percentages of mono-unsaturated and polyunsaturated fatty acids were higher in oocytes and granulosa cells during the winter. Oocytes and granulosa cells had similar fatty acid compositions, in contrast to follicular fluid. These results may explain the differences in the ability of oocytes to develop to the blastocyst stage at different seasons. Thus, temperature changes may lead to changes in membrane properties, which, in turn, can influence oocyte function and fertility.

The fertility of dairy cows decreases during the summer and remains low during the cooler autumn although the animals are no longer under heat stress. The aim of this study was to characterize a delayed effect of summer heat stress on oocyte quality in the autumn and to improve oocyte quality by enhanced removal of follicles damaged during the previous summer. Lactating cows (n = 16) were subjected to heat stress during the summer. In autumn, ovarian follicles (3-7 mm in diameter) were aspirated by an ultrasound-guided procedure during four consecutive oestrous cycles. Follicles were aspirated from control cows on day 4 and from treated cows on days 4, 7, 11 and 15 of each oestrous cycle. All cows received PGF(2alpha) and GnRH injections on days 19 and 21, respectively, and maintained cyclicity, as indicated by plasma progesterone concentrations. On day 4 of each cycle, the oocytes recovered were examined morphologically, matured and activated in vitro, and cultured for 8 days. In cycle 1 (early October) both groups showed low percentages of grade 1 oocytes, cleavage, four- and eight-cell embryos, morulae and parthenogenetic blastocysts. Subsequently, the number of grade 1 oocytes increased earlier (cycle 2) in treated than in control cows (cycle 3; P < 0.05). The cleavage rate in the control group remained relatively low throughout (32-58%), whereas in the treated group it increased from 40% (cycle 1) to 75% (cycles 3 and 4; P < 0.05). The number at each stage of embryo development increased slightly but remained low throughout in the control group, whereas in the treated group significant (P < 0.05) increases of all stages were observed in cycles 3 and 4. The results show a delayed effect of summer heat stress on oocyte quality and embryo development in the autumn. Enhanced removal of the impaired cohort of follicles led to earlier emergence of healthy follicles and high quality oocytes in the autumn.