To evaluate pregnancy outcome of frozen embryo transfer, compared to fresh embryo transfer between two groups of patients; advanced reproductive age (≥35 yo) vs. young reproductive age (<35 yo).

To investigate the effects of culture media with different lactate concentrations on early embryonic development, data collected from our patients undergoing preimplantation genetic testing (PGT) were assessed using the EmbryoScope™ time-lapse culturing system. After intracytoplasmic sperm injection (ICSI), sibling oocytes were cultured in the same EmbryoScope (Vitrolife) slides including two different commercially available media. The patients with fewer than five mature oocytes were not included in the analyses. All embryos were hatched on day 3, and trophectoderm biopsies (n = 212) were performed accordingly. PGT for aneuploidy (PGT-A) on biopsied materials was carried out using next generation sequencing. Morphokinetic parameters, fertilization, irregular division, degeneration, blastulation, euploidy, and pregnancy rates of embryos cultured in LifeGlobal Global Total medium (LGGT) and Continuous Single Culture-NX Complete medium (CSCM-NXC) were compared. There were no differences observed in time to pronuclear fade, or in time spent as 2-cell (cc2) and 3-cell (s2), to 4-cell, 5-cell, morula and blastocyst stages (P > 0.05). Embryos reached the 2-cell (t2) and 3-cell (t3) stages significantly faster in LGGT (P < 0.05), whereas embryos grown in CSCM-NXC with lower lactate reached starting blastulation significantly sooner (P = 0.026). However, there were no statistical differences observed in fertilization, blastulation, degeneration, irregular division euploidy, and pregnancy rates between the two groups (P > 0.05). Even though pregnancy and fertilization rates did not indicate statistical differences, results are significant to provide better insight on potential roles of lactate in embryo development. These finding will advance the fundamental knowledge of human embryo development and assisted reproductive technologies.

Low fertility is the single most important factor limiting livestock reproductive performance, adversely affecting the cattle industry and causing millions of dollars of economic loss. In the livestock industry, male fertility is of crucial importance for the reproductive performance of livestock. However, there is a lack of reliable biomarkers to predict bull fertility in artificial insemination service. The objective of this study was to identify sperm proteins as biomarkers for bull fertility. To discover candidate sperm quality biomarkers, sperm proteome profiling was conducted in extreme high- and extreme low-fertile bulls selected from a pool of 1000 AI sires with varied fertility. Thirty-two differentially expressed proteins were identified. Among them, high levels of sperm outer dense fiber of sperm tails 2 (ODF2) and post-acrosomal assembly of sperm head protein (PAWP/WBP2NL) represented the most extreme differences in quantity between high- and low-fertility bulls. Protein immunodetection and flow cytometry used to validate these putative fertility markers in a combined cohort of 154 AI sires. Both ODF2 and PAWP correlated significantly with fertility. In conclusion, ODF2 and PAWP can be used to assess semen quality and predict sire fertility.

The goal of the study was to ascertain sperm proteins as fertility markers by identifying sperm proteins in Angus bull sperm using proteomics and validate the markers through comparative sperm biology between Angus and Holstein bulls for which there is reliable fertility data available. We aimed to determine proteins differentially expressed in sperm from Angus bulls with different fertility phenotypes. Two-dimensional differential gel electrophoresis with mass-spectrometry, functional gene clusters, canonical pathways and protein networks, using integrated discovery bioinformatics software and ingenuity pathway analysis were used to identify and analyze sperm proteome. We identified 80 proteins that were differentially expressed in sperm of our experimental population. Using computational biology approaches we demonstrated involvement of structural proteins such as outer dense fiber of sperm tails 2 and enzymes including kinases, and phosphatases having functions in essential pathways in glycolysis/gluconeogenesis and free scavenging. The results are significant because analyzed proteins in Angus sperm are determinants of fertility, gene-environment interactions, as well as potential biomarkers for animal breeding.

To investigate the morphokinetic effects of culture media in early embryonic development. Retrospective randomized study. Data used in this study were collected from our routine IVF-PGT patients who used either autologous and/or donor oocytes between February and December 2018. All cases whose embryos were incubated in conventional incubators were excluded. Emrbyoscope slides were prepared using two different media where well 1-6 contained one media vs. well 7-12 had the second media and equilibrated overnight. On the day of retrieval, patients with at least 10 MII oocytes were randomly selected for this study. After ICSI, MII oocytes (n=35 patients, n=404 MII oocytes) were divided into two groups, and cultured in the same embryoscope (Vitrolife) slides including two different media; Group #1 (Global total by Life global) vs Group #2 (Low lactate: 1mM, CSCM-NX by Irvine). The embryos were hatched on Day 3 and trophectoderm biopsies (n=212) were performed accordingly. The biopsied materials were sent to CooperGenomics for Next Generation Sequencing [NGS] testing. There was no bias between two groups in this study because, the sibling oocytes were incubated under the same condition. Morphokinetic parameters were analysed using t-test, Fertilization, Irregular division, Blastulation and Euploidy rates were analysed using χ2-test among the groups. According to our findings, all results were shown in Table 1. The differences were considered statistically significant once p-values are < 0.05.Tabled 1GroupsGroup 1Group 2p-valueNumber of MII202202N/AtPB23.6 ± 0.93.9 ± 1.10.15tPNa7.2 ± 1.27.8 ±2.30.2tPNf24.6 ±3.325.3 ±2.60.12t226.9 ±3.729.0 ±4.10.01*t337.0 ±4.438.7 ±4.30.02*t439.7 ±4.640.9 ±5.50.07t550.0 ±5.451.1 ±6.20.31cc2 (t3-t2)9.7 ±3.49.9 ±2.30.68s2 (t4-t3)2.3 ±2.51.8 ±3.10.44tM95.0 ±8.094.6 ± 9.00.84tSB105.0 ±8.3102.2 ± 8.30.03*tB111.4 ±9.7109.9 ± 8.30.38tEB117.9 ±8.4119.2 ± 10.60.31Fertilization (%)81.80%80%0.68Euploidy (%)36%42%0.09Aneuploidy (%)45%31%0.11Mosaicism (%)17%22%0.11Irregular Division (%)17%21%0.27Day 5 Blastulation (%)58%52%0.38 Open table in a new tab In this study, we only demonstrated that the t2 and t3 divisions were earlier, and blastulation was later in Group #1 than Group #2. Although the euploidy rate was higher in media with low lactate (Group #2), this difference was not statistically significant. In conclusion, a larger sample size is needed to conclude the positive effects of culture media with low lactate.

Objective To determine if morphokinetic parameters differ between euploid and aneuploidy emryo development using time-laps imaging (TLI) and preimplantation genetic screening (PGS). Design Retrospective chart review Materials and Methods A total of 613 embryos from 128 patient couples were evaluated for ploidy status and morphokinetic time points using TLI January,2016- March,2017 were analyzed for TLI, ploidy status and infertility diagnosis. The subject population included couples going through IVF using TLI culture and PGS testing by NGS or aCGH. Important morphokinetic cell divisions were annotated: 2-9+ cell stages annotated as t2, t3, t4, t5, t6, t7, t8 and t9+ and morula formation), starting blastulation(tSB), expanding blastocyst(tEB), and hatching blastocyst(tHB) determined. The Chi square test was used to detect associations between ploidy status,age,infertility diagnosis and PGS results. Two sample student t test was used to compare the difference in average morphokinetic outcomes, t2-t8, between euploid and aneuploid embryos. Cell cycle time between the 2 and 3 cell stage (CC2) and between the 3 and 4 cell stage (S2) were calculated. As these differences lack the normality assumption, we analyzed the difference between CC2 and S2 by using Wilcoxon-Mann Whitney U test to compare between aneuploidy and euploid PGS results. Diagnosis comparisons displayed as descriptive statistics. Results There were no significant differences in most morphokinetic time points between euploid and aneupliod embryos. There was a statistically significant difference between euploid and aneuploid development in the CC2 (-1.088±7.093; p-value= 0.0404) as well as a marginal difference between euploid and aneuploid embryos is S2 (0.963±0.0512; p-value= 0.0512) . It is noticed that ploidy status significantly different between female ≤ 34 yrs. and > 34 yrs. of age (p-value=0.043). The odds of euploid among female ≤ yrs. is 1.465 times compared to that of females > 34 yrs. old. Therefore, younger females are 47% more likely to have euploid embryos compared to older women. Conclusions The observation and comparison of kinetic behavior through TLI and morphokinetic calculations of CC2 and S2 can aid in prediction of embryo ploidy status in combination with PGS test results. To determine if morphokinetic parameters differ between euploid and aneuploidy emryo development using time-laps imaging (TLI) and preimplantation genetic screening (PGS). Retrospective chart review A total of 613 embryos from 128 patient couples were evaluated for ploidy status and morphokinetic time points using TLI January,2016- March,2017 were analyzed for TLI, ploidy status and infertility diagnosis. The subject population included couples going through IVF using TLI culture and PGS testing by NGS or aCGH. Important morphokinetic cell divisions were annotated: 2-9+ cell stages annotated as t2, t3, t4, t5, t6, t7, t8 and t9+ and morula formation), starting blastulation(tSB), expanding blastocyst(tEB), and hatching blastocyst(tHB) determined. The Chi square test was used to detect associations between ploidy status,age,infertility diagnosis and PGS results. Two sample student t test was used to compare the difference in average morphokinetic outcomes, t2-t8, between euploid and aneuploid embryos. Cell cycle time between the 2 and 3 cell stage (CC2) and between the 3 and 4 cell stage (S2) were calculated. As these differences lack the normality assumption, we analyzed the difference between CC2 and S2 by using Wilcoxon-Mann Whitney U test to compare between aneuploidy and euploid PGS results. Diagnosis comparisons displayed as descriptive statistics. There were no significant differences in most morphokinetic time points between euploid and aneupliod embryos. There was a statistically significant difference between euploid and aneuploid development in the CC2 (-1.088±7.093; p-value= 0.0404) as well as a marginal difference between euploid and aneuploid embryos is S2 (0.963±0.0512; p-value= 0.0512) . It is noticed that ploidy status significantly different between female ≤ 34 yrs. and > 34 yrs. of age (p-value=0.043). The odds of euploid among female ≤ yrs. is 1.465 times compared to that of females > 34 yrs. old. Therefore, younger females are 47% more likely to have euploid embryos compared to older women. The observation and comparison of kinetic behavior through TLI and morphokinetic calculations of CC2 and S2 can aid in prediction of embryo ploidy status in combination with PGS test results.

Objective: To investigate the effects of sperm concentration and motility on embryo morphokinetics and ploidy status.

ObjectiveTo investigate the differences in euploidy rates between PGS techniques in patients undergoing blastomere vs trophectoderm biopsy.DesignRetrospective cohort study.Materials and MethodsData used in this study were collected from our routine PGS patients who used either autologous and or donor oocytes from 2014 to 2016 for incubation following insemination (n=365). Translocation and PGD cycles were excluded. The resulting embryos were divided into four groups based on biopsy day (blastomere versus trophoectoderm) and PGS technique used (comparative genomic hybridization microarray [aCGH] vs Next Generation Sequencing [NGS]) (n=2010): Group 1 (aCGH blastomere, n=873), Group 2 (aCGH trophectoderm, n=239), Group 3 (NGS blastomere, n=21) and Group 4 (NGS trophectoderm, n=877). The use of oocyte donor was similar between groups. Patients' ages were ranged as following; Group 1 (20-48), Group 2 (25-46), Group 3 (27-36) and Group 4 (23-47). Euploidy rates were analysed using chi square test among the groups.ResultsAccording to our findings, euploidy rates (n) were found to be 22.1% (193) and 32.6% (78) (p< .001) within Group 1 vs Group 2, respectively. Likewise, the euploidy rates were different in Group 3 as 23.8% (5) and Group 4 as 47% (412), respectively (p< .035). The euploidy rate in Group 4 was higher than that in Group 2 (p<.00001). The results were shown as a table below. There was no difference between Group 1 vs Group 3 (p>.05).ConclusionsTabled 1Euploidy Rates of Embryos within GroupsGroup 1Group 2Group 3Group 422.1%32.6%23.8%47% Open table in a new tab ObjectiveTo investigate the differences in euploidy rates between PGS techniques in patients undergoing blastomere vs trophectoderm biopsy. To investigate the differences in euploidy rates between PGS techniques in patients undergoing blastomere vs trophectoderm biopsy. DesignRetrospective cohort study. Retrospective cohort study. Materials and MethodsData used in this study were collected from our routine PGS patients who used either autologous and or donor oocytes from 2014 to 2016 for incubation following insemination (n=365). Translocation and PGD cycles were excluded. The resulting embryos were divided into four groups based on biopsy day (blastomere versus trophoectoderm) and PGS technique used (comparative genomic hybridization microarray [aCGH] vs Next Generation Sequencing [NGS]) (n=2010): Group 1 (aCGH blastomere, n=873), Group 2 (aCGH trophectoderm, n=239), Group 3 (NGS blastomere, n=21) and Group 4 (NGS trophectoderm, n=877). The use of oocyte donor was similar between groups. Patients' ages were ranged as following; Group 1 (20-48), Group 2 (25-46), Group 3 (27-36) and Group 4 (23-47). Euploidy rates were analysed using chi square test among the groups. Data used in this study were collected from our routine PGS patients who used either autologous and or donor oocytes from 2014 to 2016 for incubation following insemination (n=365). Translocation and PGD cycles were excluded. The resulting embryos were divided into four groups based on biopsy day (blastomere versus trophoectoderm) and PGS technique used (comparative genomic hybridization microarray [aCGH] vs Next Generation Sequencing [NGS]) (n=2010): Group 1 (aCGH blastomere, n=873), Group 2 (aCGH trophectoderm, n=239), Group 3 (NGS blastomere, n=21) and Group 4 (NGS trophectoderm, n=877). The use of oocyte donor was similar between groups. Patients' ages were ranged as following; Group 1 (20-48), Group 2 (25-46), Group 3 (27-36) and Group 4 (23-47). Euploidy rates were analysed using chi square test among the groups. ResultsAccording to our findings, euploidy rates (n) were found to be 22.1% (193) and 32.6% (78) (p< .001) within Group 1 vs Group 2, respectively. Likewise, the euploidy rates were different in Group 3 as 23.8% (5) and Group 4 as 47% (412), respectively (p< .035). The euploidy rate in Group 4 was higher than that in Group 2 (p<.00001). The results were shown as a table below. There was no difference between Group 1 vs Group 3 (p>.05). According to our findings, euploidy rates (n) were found to be 22.1% (193) and 32.6% (78) (p< .001) within Group 1 vs Group 2, respectively. Likewise, the euploidy rates were different in Group 3 as 23.8% (5) and Group 4 as 47% (412), respectively (p< .035). The euploidy rate in Group 4 was higher than that in Group 2 (p<.00001). The results were shown as a table below. There was no difference between Group 1 vs Group 3 (p>.05). ConclusionsTabled 1Euploidy Rates of Embryos within GroupsGroup 1Group 2Group 3Group 422.1%32.6%23.8%47% Open table in a new tab

Objective: To investigate the gender-specific morphokinetics of embryos. Design: Retrospective observational study. Materials and Methods: Data used in this study were collected from our routine IVF-PGS patients during 2013–2015, excluding donor, frozen and PGD cycles. Autologous fresh oocytes from randomly selected patients (n = 65; 69 cycles) were incubated in Time-Lapse microscope (EmbryoScope: Unisense Fertilitech, Denmark) following insemination. Following the biopsy either on Day 3 or Day 5 (n = 358), ploidy status of embryos was analyzed by Genesis Genetics. According to PGS results, these embryos classified into two groups based on their gender after excluding embryos with complex abnormality on multiple chromosomes (n = 96) and no result (n = 8). The gender specific morphokinetic parameters and the most common chromosomal abnormalities were determined in XX embryos (n = 125) vs. XY embryos (n = 129) (Table 1). Data were analyzed using student t-test and χ2-test. Results: The mean differences of all parameters within groups were tabulated below.Tabled 1XX EMBRYOSXY EMBRYOSP-VALUENUMBER OF EMBRYOS125129AGE35.7 ± 5.135.8 ± 5.00.78TPNA (TIME OF PN APPEARANCE)9 ± 2.99.5 ± 3.10.22T2 (TIME OF 2ND BLASTOMERE APPEARANCE)27.2 ± 7.627.6 ± 6.70.74T3 (TIME OF 3RD BLASTOMERE APPEARANCE)37.7 ± 7.437.7 ± 7.10.95CC2 (TIME OF 2ND CELL CYCLE; T3-T2)10.4 ± 4.510.1 ± 4.20.51T4 (TIME OF 4TH BLASTOMERE APPEARANCE)39.8 ± 7.740.0 ± 6.90.93S2 (TIME OF SYNCHRONY OF 2ND CELL CYCLE; T4-T3)2.1 ± 4.32.3 ± 4.50.80T5 (TIME OF 5TH BLASTOMERE APPEARANCE)51.6 ± 8.051.8 ± 8.30.95CC3 (TIME OF 3RD CELL CYCLE; T5-T3)13.9 ± 5.912.9 ± 10.60.32T8 (TIME OF 8TH BLASTOMERE APPEARANCE)61.2 ± 9.462.7 ± 9.40.25TM (TIME FROM INSEMINATION TO FORMATION OF A MORULA)90.2 ± 10.790.6 ± 11.30.83EUPLOID (%)62 (49.6%)72 (55.8%)0.16ANEUPLOID (%)63 (50.4%)57 (44.2%)0.16MONOSOMY (%)20 (31.7%)16 (28.1%)0.21TRISOMY (%)15 (23.8%)17 (29.8%)0.39CHROMOSOME 16 (%)9 (14.3%)2 (3.5%)0.01*CHROMOSOME 21 (%)4 (6.3%)6 (10.5%)0.28 Open table in a new tab Conclusions: Our findings showed that the morphokinetics of embryos were similar between groups. Chromosome 16 is mostly affected in female embryos compared to their male counterparts*. A detailed chromosome screening such as SNP studies might reveal better understating of gender-specific characteristics of embryos. Disclosures: Nothing to disclose. Funding: None.

During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin structure, coinciding with reduced fertility in bulls. These findings are highly significant because they reveal molecular and morphological phenotypes of mammalian spermatozoa that influence fertility.

cant. RESULTS: In the implanted group, 21(51.2%) embryos were observed withmononucleationat2-cellstage,while15(36.6%)presented1-cellmultinucleation (C1), 5 (12.2%) presented both cells multinucleation(C2), and 5 (12.2%) presented RC. In the non-implanted group, 25(50%) embryos were observed with mononucleation at 2-cell stage, while 14 (28%) presented C1, 11 (22%) presented C2, and 6 (12%) presented RC. Comparisons of 2-cell stage mononucleation and multinucleation rates between implanted and non-implanted were not significant. The implantation rates of mononucleation, C1, and C2 were 45.7%, 51.7%, and 31.3% respectively. The implantation rates of RC and non-RC groups were 45.5 % and 45%. The differences between these implantation rates were not significant, either. CONCLUSION: Blastocysts from mono and multinucleated 2-cell embryos have equal chances of implantation success. Reverse cleavage is not a significant factor in unsuccessful implantation.

To re-investigate the morphokinetic characteristics of aneuploidy in embryos from randomly selected IVF-PGS (preimplantation genetic screening) patients whose ages are normally distributed. Retrospective cohort study. Data used were from our routine IVF-PGS patients (n=18) whose ages are normally distributed [25.8-43.6]. Autologous fresh oocytes (n=185) from these patients were incubated in Time-Lapse microscope (EmbryoScope: Unisense Fertilitech, Denmark) following insemination. After Day 3 biopsy (n=152), ploidy was analyzed using array-comparative genome hybridization (aCGH). According to PGS results, these embryos classified into two groups, euploid (n=46) and aneuploid (n=106). The morphokinetic parameters tested in this study, including three new parameters* [oocyte diameter, distance of pronuclei (PNs) and ratio of PN diameters] are listed in Table 1. Data were analyzed using one way ANOVA and χ2-test, and Pearson correlation test. In this study, aeuploidy is only correlated with patient's age (r=.331, p< .0001). Within the two groups of embryos, the mean differences for the tested morphokinetic parameters are tabulated below.Tabled 1The Mean Differences of Morphokinetic Parameters within GroupsParametersEuploidyAneuploidyP-valueAge34.1 ±4.237.5 ±4.60.000**Oocyte diameter*159.3 ±11.0159.6 ±7.40.835Distance of PNs*44.2 ±5.049.7 ±49.50.462Ratio of PN diameters*1.1 ±0.21.0 ±0.20.386Granular cytoplasm10.9%11.3%0.935tPB2 (time of polar body extrusion)4.7 ±3.34.8 ±3.40.810tPNa (time of PN appearance)10.5 ±3.510.8 ±3.90.627tPNf (time of PN faded)24.2 ±4.624.6 ±6.50.686cc2 (time of 2nd cell cycle; t3-t2)7.5 ±5.87.8 ±6.50.673s2 (time of synchrony of 2nd cell cycle; t4-t3)4.5 ±5.25.4 ±5.50.308cc3 (time of 3rd cell cycle; t5-t3)15.0 ±7.114.9 ±6.70.954tM (time from insemination to formation of a morula)89.6 ±12.389.6 ±12.40.984tSB (time from insemination to start of blastulation)100.6 ±11.699.9 ±8.90.750tB (time from insemination to formation of a full blastocyst)106.7 ±8.9106.9 ±8.00.925PN score grade I+II80%88.6%0.332Irregular Division10.9%21.7%0.114Recleavage8.7%6.6%0.647Multinucleation at 2 cell stage32.6%36.8%0.621Blastomere size (even)37.0%33.0%0.268 Open table in a new tab The results here revealed that aneuploidy cannot be predicted using the morphokinetic parameters tested by time-lapse microscopy without PGS screening [1]. Patient's age in the PGS cases should also be considered as a parameter for further time-lapse studies.

To investigate if nuclear status at 2 cell stage and reverse cleavage between 2∼4-cell stage of transferred embryo, are related to implantation outcome. Retrospective study. After Intracytoplasmic sperm injection, the metaphase II oocytes were cultured in EmbryoScope (Unisense) time-lapse system. Transferred embryos were retrospectively analyzed in EmbryoViewer database. Ninety-one transferred embryos from 52 women were analyzed. Forty-one implanted transferred embryos were from 25 patients and 50 non-implanted from 27 patients. The presence of multinucleation at 2-cell stage and reverse cleavage were compared between positive and negative implanted groups. Reverse cleavage (RC) was defined as a decrease in the number of cells during cytokinesis. Namely, cells in blastomere fused together to form a blastomere with lesser cells, and they cleaved again after that. This phenomenon was specifically observed with time-lapse imaging system, and noticed previously [1]. Implantation was assessed by ultrasound in 4 weeks after embryo transfer. Double embryo transfers with only one heart beat were excluded. Data were analyzed using χ2-test and P-value < 0.05 was considered significant. In the implanted group, 21(51.2%) embryos were observed with mononucleation at 2-cell stage, while 15 (36.6%) presented 1-cell multinucleation (C1), 5 (12.2%) presented both cells multinucleation(C2), and 5 (12.2%) presented RC. In the non-implanted group, 25(50%) embryos were observed with mononucleation at 2-cell stage, while 14 (28%) presented C1, 11 (22%) presented C2, and 6 (12%) presented RC. Comparisons of 2-cell stage mononucleation and multinucleation rates between implanted and non-implanted were not significant. The implantation rates of mononucleation, C1, and C2 were 45.7%, 51.7%, and 31.3% respectively. The implantation rates of RC and non-RC groups were 45.5 % and 45%. The differences between these implantation rates were not significant, either. Blastocysts from mono and multinucleated 2-cell embryos have equal chances of implantation success. Reverse cleavage is not a significant factor in unsuccessful implantation.

Sub-par fertility in bulls is influenced by alterations in sperm chromatin, and it might not be solved with increased sperm concentration in artificial insemination. Appropriate histone retention during sperm chromatin condensation plays critical roles in male fertility. The objective of this study was to determine failures of sperm chromatin condensation associated with abnormal persistence or accessibility of histones by aniline blue (ANBL) test, expression levels, and cellular localizations of one variant and two core histones (H3.3, H2B, and H4 respectively) in the spermatozoa of low-fertility (LF) vs high-fertility (HF) bulls. The expression levels and cellular localizations of histones in spermatozoa were studied using immunoblotting, immunocytochemistry, and staining methods. The bioinformatics focused on the sequence identity and evolutionary distance of these proteins among three mammalian species: bovine, mouse, and human. We demonstrated that ANBL staining was different within the LF (1.73 (0.55, 0.19)) and HF (0.67 (0.17, 0.06)) groups (P<0.0001), which was also negatively correlated with in vivo bull fertility (r=-0.90, P<0.0001). Although these histones were consistently detectable and specifically localized in bull sperm cells, they were not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and were evolutionarily conserved in bulls, mice and humans. The H2B variants were more conserved between bulls and humans, than in mice. In conclusion, we showed that H2B, H3.3, and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related to bull fertility.

Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (+/- 1.31) and 3.00 (+/- 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r=-0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r=-0.49, P<0.006 and r=-0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1-3 vs. Bull 4-5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.

Fertilization of an egg by a spermatozoon sets the stage for mammalian development. Viable sperm are a prerequisite for successful fertilization and beyond. Spermatozoa have a unique cell structure where haploid genomic DNA is located in a tiny cytoplasmic space in the head, mitochondria in the midpiece and then the tail, all enclosed by several layers of membrane. Proteins in sperm play vital roles in motility, capacitation, fertilization, egg activation and embryo development. Molecular defects in these proteins are associated with low fertility or in some cases, infertility. This review will first summarize genesis, molecular anatomy and physiology of spermatozoa, fertilization, embryogenesis and then those proteins playing important roles in various aspects of sperm physiology.

Genomic DNA of bull spermatozoa is tightly packaged mostly around Protamine 1 (PRM1) and small amounts of histones such as histone3 (H3) in a much smaller volume compared to somatic cells. Proper packaging of genome is important both for sperm morphology and physiology as well as for fertilization and embryonic development. Both PRM1 and histone3.3 (H3.3; a variant of H3) are known to play important roles during sperm chromatin condensation. Recently, H3.3 has been shown to be bound to actively transcribed regions of the paternal genome; therefore, its presence becomes important. However, molecular and cellular underpinnings of their functions in bull spermatozoa and how these two proteins regulate fertility are not known. The objective of this study was to determine the dynamics of chromatin structure and the expression patterns of DNA binding proteins PRM1 and H3.3 in spermatozoa from bulls with varying fertility. To examine sperm chromatin condensation, chromatin integrity and protamination were detected using halomax assay and toluidine blue staining in spermatozoa from 20 bulls with distinct fertility, which was equally grouped into low and high fertility. For both methods, three biological and two technical replicates were performed per bull, and 500 spermatozoa were counted per slide by two different technicians using light microscopy. We also performed western blotting to detect expression levels of PRM1 and H3.3 followed by acetic acid-urea gel system and computational biology to determine functional motifs in the two proteins. In addition, spermatozoa from three bulls per group were used to conduct IVF experiments with three replicates. Statistical analysis was performed using SAS. Our results revealed that sperm chromatin integrity 7.01% (±2.24) vs. 3.97% (±0.95); p < 0.0001 and protamination 3.27% (±0.99) vs.1.2% (±0.91); p < 0.0001 were significantly different between low and high fertility groups, respectively. Based on the IVF results, embryo cleavage and development rates to blastocyst stage were lower in low fertility group compared to their high fertility counterparts 57.56% (±10.21) vs. 68.92% (±11.65); p <0.05 and 11.7% (± 3.50) vs. 16.96% (± 6.51); p < 0.05, respectively. We confirmed that slide readings of halomax and toluidine blue experiments were not different between the two technicians (p > 0.54 and > 0.80, respectively). We also found that in vivo fertility scores of the bulls were negatively correlated with sperm chromatin protamination (r= −0.62; p < 0.0001) and integrity (r= −0.69; p < 0.0001). In addition, sperm chromatin integrity among the bulls was negatively associated with embryo cleavage (r= −0.56; p < 0.05) and development rate (r= −0.45; p = 0.0637) in embryos derived using spermatozoa from the bulls with varying fertility. In contrast to PRM1, functional motifs were only found in H3 such as histone H3.2, CAMP phospho site, myristyl and Ck2 phospho. We also showed that there is a ratio between the protein expression levels of PRM1 and H3.3 among the bulls. In conclusion, our data showed that inadequate sperm chromatin protamination and integrity were associated with inefficient sperm chromatin condensation leading to improper fertilization and beyond, which can be estimated using bull fertility scores prior to use them for AI. Our results provide a comprehensive output in sperm chromatin dynamics that impacts sperm viability, fertilization and early embryonic development.