Conventional Semen Research Articles

Association between semen quality among men with different occupational exposures and risk of recurrent spontaneous abortion in island residents

Objective: This study aimed to evaluate the semen quality of male adults whose spouses suffer from recurrent spontaneous abortion (RSA) in the island area, and to explore the association between RSA and occupational exposures. Methods: A total of 131 male patients were recruited and divided into two groups: spouse with recurrent spontaneous abortion group (RSA group, [Formula: see text]) and the normal fertility group (control group, [Formula: see text]). Information such as height, weight and occupational exposure history of 131 men were obtained. Semen samples were collected and analyzed. Differences in semen parameters and DNA fragmentation index (DFI) between the two groups were compared. Odds ratios (ORs) and their corresponding 95% confidence intervals were calculated to evaluate the association between occupational exposures and RSA status. Results: Overall, no significant difference was found in sperm concentration, progressive motility and normal morphology rate between RSA and control groups. Only DFI was observed to be significantly higher in the RSA group. Evaluation of the receiver operating characteristic (ROC) curve showed DFI (AUC: 0.623, [Formula: see text]) could discriminate between males from the couples with RSA and without RSA. Conclusion: The analysis of conventional semen parameters could not directly reflect their influence on embryonic development, and test of integrity of the sperm DNA is of paramount importance to fully understand male fertility. It is crucial to conduct studies regarding occupational exposures and pregnancy loss and/or RSA risk, since it will provide population-level data to aid in the identification of important risk factors that warrant further mechanistic investigation, and eventually lead to effective prevention and treatment strategies.

Effects of intrauterine infusion of seminal plasma at artificial insemination on fertility of lactating Holstein cows

An inflammatory response is induced in the reproductive tract by deposition of semen during natural mating. This response might facilitate establishment and maintenance of pregnancy and alter the phenotype of the offspring by modifying the microenvironment of the reproductive tract. Here, we hypothesized that intrauterine infusion of 0.5 mL of seminal plasma at the time of artificial insemination (AI) in first-service lactating Holstein cows will improve pregnancy success after insemination. Cows were inseminated (511 primiparous cows inseminated with X-sorted semen, 554 multiparous cows inseminated with X-sorted semen, and 627 multiparous cows inseminated with conventional semen) using the Double-Ovsynch protocol. Cows were randomly assigned to receive intrauterine infusion of either 0.5 mL of seminal plasma or saline immediately after AI. There was no overall effect of seminal plasma infusion on the percentage of inseminated cows diagnosed pregnant at d 32 or 60 after AI, pregnancy loss, or percent of inseminated cows calving. If cows were inseminated with conventional semen, seminal plasma reduced pregnancies at d 32 and tended to reduce calvings. There was no effect of seminal plasma if cows were inseminated with X-sorted semen. Seminal plasma infusion increased the birth weight of heifer calves born using X-sorted semen but not conventional semen. These results do not support a beneficial effect of seminal plasma on pregnancy success after AI, but exposure to seminal plasma may program fetal development to affect phenotype at birth.

DNA fragmentation in concert with the simultaneous assessment of cell viability in a subfertile population: establishing thresholds of normality both before and after density gradient centrifugation.

TUNEL assay is the most common, direct test for sperm chromatin integrity assessment. But, lack of standardized protocols makes interlaboratory comparisons impossible. Consequently, clinical thresholds to predict the chance of a clinical pregnancy also vary with the technique adopted. This prospective study was undertaken to assess the incidence of sperm DNA fragmentation in a subfertile population and to establish threshold values of normality as compared to a fertile cohort, both before and after density gradient centrifugation in the total and vital fractions. Men presenting at a university hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. Total DNA fragmentation in the neat semen was significantly higher in the subfertile group, but the vital fraction was not significantly different between the two cohorts. After gradient centrifugation, DNA fragmentation increased significantly in the total fraction of the subfertile group but decreased significantly in the vital fraction. In the fertile cohort, there was a non-significant increase in total fragmentation and in the vital fraction the trend was unclear. Estimating total and vital sperm DNA fragmentation, after density gradient centrifugation, increased both the sensitivity and the specificity, thereby lowering the number of false negatives and false positives encountered. These findings provide opportunities to investigate the significance of the total and the vital fractions after different assisted reproductive technologies.

Sperm DNA fragmentation valued by SCSA and its correlation with conventional sperm parameters in male partner of recurrent spontaneous abortion couple.

The objective of this study is to evaluate the predictive value of sperm DNA fragmentation Index (DFI) in unexplained recurrent spontaneous abortion (RSA) and to investigate its correlation with conventional sperm parameters. Besides, we aimed to reveal the necessity of establishing a DFI clinical threshold of each laboratory for the prognostic diagnosis of RSA and establish our own DFI threshold. Semen samples were collected from male partners of RSA patients (n = 139) and healthy recent fathers (control, n = 200). DFI was tested using SCSA and conventional semen analysis was performed using an automatic semen analyzer. The DFI value and distribution were compared between the two groups using corresponding statistical software. The diagnostic threshold value was established by ROC curve. The correlation between DFI and the conventional semen parameters of the 139 cases was further analyzed using Student's t test and Mann-Whitney U test. Our result showed that DFI was significantly higher in RSA patients compared with normal donor controls. We established our own DFI threshold at 13.59%. There was only a weak partial correlation between DFI values and conventional sperm analysis parameters. Our present study suggested that DFI might be used as a valuable predictor for RSA independent of conventional sperm parameters. Additionally, we recommend that each laboratory should establish its own clinical DFI threshold for more precise prediction of RSA and we recommend that sperm DNA fragmentation test should be included in complete sperm quality assessment in addition to conventional semen analysis for RSA male partners.

Effect of presynchronization with prostaglandin F2α before the 5-d timed AI protocol on ovarian responses and pregnancy in dairy heifers

The objectives were the determine the effects of presynchronization with PGF2α 2 days before the 5-d timed artificial insemination (AI) protocol on ovarian responses and pregnancy per AI (P/AI) in dairy heifers. The hypothesis was that PGF2α would induce responsive heifers to be in proestrus at the initiation of the timed AI protocol, which was expected to improve ovulatory responses and P/AI. Weekly cohorts of Holstein heifers were blocked by age and, within block, randomly assigned to remain as control (CON; n = 255) or receive PGF2α on experiment Day −10 (PG; n = 255). All heifers were subjected to the 5-d timed AI protocol (Day −8, GnRH + intravaginal progesterone controlled internal drug release insert; Day −3, PGF2α and insert removal; Day −2, PGF2α; and Day 0, GnRH and AI). A subset of 22 blocks of heifers (n = 43) had their ovaries scanned by ultrasonography on experiment Days −8, −3, 0, and 2 and blood was sampled and analyzed for concentrations of progesterone on experiment Days −8, −7, −5, and −3. Pregnancy was diagnosed on experiment Days 32 and 60. On the day of the first GnRH of the timed AI protocol, PG heifers had smaller concentration of progesterone in plasma (CON = 4.5 ± 0.5 vs. PG = 0.5 ± 0.5 ng/mL), but larger follicular diameter (CON = 9.1 ± 0.5 vs. PG = 11.0 ± 0.5 mm), and a greater proportion of them had a follicle with at least 8.0 mm in diameter (CON = 61.9 vs. PG = 90.9%) than CON heifers, which resulted in increased ovulation to GnRH (CON = 19.0 vs. PG = 86.3%). Ovulation to the initial GnRH of the protocol increased as the concentration of progesterone in plasma decreased, from less than 20% when progesterone was greater than 5.0 ng/mL to more than 65% when progesterone was less than 1.0 ng/mL. More CON than PG heifers spontaenously ovulated before the day of timed AI. Detection of estrus on the day of timed AI did not differ between treatments (CON = 50.9 vs. PG = 46.6%), but P/AI on Days 32 (CON = 52.9 vs. PG = 61.1%) and 60 (CON = 49.0 vs. PG = 57.1%) after insemination tended to be greater for PG than CON; however, the benefit to presynchronization was observed in heifers inseminated with conventional (CON = 54.7 vs. PG = 67.4%), but not in heifers inseminated with sex-sorted semen (CON = 50.9 vs. PG = 52.8%). Administration of PGF2α 2 days before initiating the timed AI protocol induced heifers to be in proestrus, which enhanced ovulation to the initial GnRH and favored pregnancy per AI, particularly in heifers inseminated with conventional semen.

Hyaluronan bound mature sperm count (HB-MaSC) is a more informative indicator of fertility than conventional sperm parameters: Correlations with Body Mass Index (BMI)

The relationship between overweight and male fertility is well studied, still the correlation of obesity and decreased sperm quality is a subject to debate. The widely used conventional spermatological examinations alone seem to be inadequate to assess fertilization potential. Hyaluronan Binding Assay (HBA®) is one of the available validated tests that allows the functional examination of sperm. Data of 72 male patients (mean age 33.9 (24–43) years) from infertile couples were analysed. Body Mass Index (BMI) determination, conventional semen analysis and HBA were performed. Additionally, a relatively new Hyaluronan Bound Matured Sperm Count (HB-MaSC) -index, first introduced by the authors in 2015, was calculated. This index reflects fertilization potential of sperm more precisely. With the increase of BMI, sperm count decreased significantly until about 25 kg/m2, above 25 kg/m2 no further decrease was observed, although sperm count remained permanently low. Greater body weight (in the 70–90 kg range) was observed to have a significant negative effect only on the progressive sperm motility. In addition to sperm concentration and motility, sperm fertilization potential is also negatively affected by obesity, but is irrespective of body weight, as evaluated using BMI + HB-MaSC linear regression analyses adjusted for age and weight. This correlation between male BMI and sperm fertilization potential – as opposed to the conventional correlations with sperm concentration or motility – appears to provide more helpful information in the identification of real capability for fertilization.

The aging male: Relationship between male age, sperm quality and sperm DNA damage in an unselected population of 3124 men attending the fertility centre for the first time.

the aim of our study was to put forward insights to treat any possible correlation among sperm quality, sperm DNA damage and male age as they may have fertility implications for men who choose to delay fatherhood. Our study is a non-interventional retrospective analysis of 3124 semen samples from patients that were investigated for the conventional semen parameters. Tunel test assay was set up for the evaluation of the sperm DNA fragmentation index (DFI). We applied the Kappa index to compare both the 1999 and the 2010 World Health Organization (WHO) reference criteria to evaluate the competence of such semen parameters categorization during the standard routine of our laboratory. With regards to our findings, it is possible to underline a significant relationship between aging and semen volume (p = 0.001), motility (p = 0.009), semen viscosity (p < 0.003) and sperm DNA damage (p < 0.009). We found a trend when focusing on the semen concentration (p = 0.05). The analysis of sperm morphology did not show any influence with advancing age (p = 0.606). When comparing both the 1999 and the 2010 WHO scales we found no accordance in the appraisal of sperm morphology but a very good one in the evaluation of the other parameters. Conventional semen analysis represents the opportunity to draw up a proxy insight on the male fertility status even if semen quality can only indirectly assess the probability of pregnancy. Several studies have verified a decay in the male reproductive system, sperm quality and fertility with advancing age although the reported results are not yet conclusive. Our results substantially agree with those findings outlined in the literature. Moreover we find that the discrepancy between the two WHO reference scales would eventually lead to an improper diagnosis of infertility.

Comparison of manual sperm analysis with computer-assisted sperm analysis: A comparative cross-sectional study

Background: Conventional manual semen analysis is done routinely in most reproductive laboratories. Computer-assisted semen analysis (CASA), on the other hand, is more objective and timesaving counterpart to the conventional method. Different CASA software systems are available, some are fully automated. Thus, it is essential to analyze its performance and compare it with the conventional semen analysis method. Aims and Objectives: Our objective was to analyze the same semen sample by manual method using Makler chamber and using sophisticated CASA software. Our aim was to find the level agreement between the two methods. Materials and Methods: Ethical clearance was taken before the study. All the subjects were given proper instructions on how to collect complete semen sample. n = 60 diagnosed primary infertile male patients referred from the obstetrics and gynecology department. Semen of the same patient was analyzed by manual method and CASA software in accordance with the WHO guidelines. The results obtained were analyzed using Pearson’s correlation and Bland and Altman plot. Results: On statistical analysis, “r” value obtained is 0.99 which is highly statistically significant. CASA and manual methods both agree with each other as proved by Bland and Altman plot. Conclusion: Both methods can be used to screen semen samples in reproductive laboratories. Manual method is easy, feasible, requires expert analysis, and has subjective variability. CASA, on the other hand, is faster newer method with better reproducibility.

Sperm DNA Integrity in Leukocytospermia and Its Association with Seminal Adenosine Deaminase.

Background:The study aimed to examine the effect of leukocytospermia on sperm quality and the levels of seminal adenosine deaminase (ADA) enzyme in males attending an infertility clinic in a tertiary hospital and to detect the association, if any, between seminal ADA and sperm quality.Methodology:Consenting male subjects, between 21 and 45 years, attending the infertility clinic and qualifying the eligibility criteria were recruited following informed consent. The collected semen samples were analyzed for the routine parameters based on the WHO protocols and for sperm DNA fragmentation. The seminal leukocyte count was detected using the peroxidase method, and the seminal ADA was assessed using spectrophotometry.Results:Samples from 110 participants were included in the study; leukocytospermia was detected in 33% of the samples. A significant reduction in the sperm quality with respect to conventional semen parameters (sperm motility and sperm vitality) and sperm DNA fragmentation index (SDFI) was noted in the presence of leukocytospermia. Furthermore, a significant positive correlation between the levels of seminal ADA and SDFI was noted (P = 0.000, r = 0.412).Conclusion:The sperm motility and DNA integrity are significantly compromised in the presence of leukocytospermia when the leukocyte count is > 1 million/mL of semen as well as 0.5–1 million/mL of semen. The positive correlation noted between seminal ADA levels and increased sperm DNA damage paves way for the possibility of seminal ADA to be an indicator of silent male genital tract inflammation as well as low-quality semen.

Reproductive performance of replacement dairy heifers submitted to first service with programs that favor insemination at detected estrus, timed artificial insemination, or a combination of both

Our objective was to compare the insemination dynamics and time to pregnancy for up to 100 d after the beginning of the artificial insemination period (AIP) for heifers managed with first artificial insemination (AI) service programs that relied primarily on insemination at detected estrus (AIE) after PGF2α treatments, timed artificial insemination (TAI), or a combination of both. Holstein heifers were randomly assigned to receive first AI service with sex-selected semen after 368 ± 10 d of age with (1) AIE after synchronization of estrus with up to 3 PGF2α treatments every 14 d starting on the first day of the AIP (PGF+AIE; n = 317). Heifers not AIE up to 9 d after the third PGF2α received a 5-d Cosynch protocol with progesterone supplementation [GnRH + controlled internal drug release insert (CIDR)-5 d-CIDR removal and PGF2α-3 d-GnRH and TAI] before TAI. Heifers detected in estrus from CIDR removal and PGF2α until the day before TAI received AIE with no GnRH treatment; (2) 2 PGF2α treatments 14 d apart with the second treatment at the beginning of the AIP (PGF+TAI; n = 334). Heifers received AIE for up to 9 d after the second PGF2α treatment. Heifers not AIE received TAI after the 5-d Cosynch protocol and (3) TAI after the 5-d Cosynch protocol (ALL-TAI; n = 315). Heifers failing to conceive to a previous AI received a subsequent AI with conventional semen at detected estrus or TAI after the 5-d Cosynch protocol. Binomial outcomes were analyzed by logistic regression, whereas time to AI and pregnancy were analyzed with Cox's regression. The hazard of first AI up to 45 d of the AIP was greater for ALL-TAI than for PGF+AIE [hazard ratio (HR) = 1.72; 95% confidence interval (CI) =1.45 to 2.03] and PGF+TAI (HR = 1.51; 95% CI = 1.28 to 1.77), but similar for PGF+AIE and PGF+TAI (HR = 1.14; 95% CI = 0.97 to 1.33). A greater proportion of heifers received AIE in PGF+AIE (98.7%) than in PGF+TAI (78.5%). Overall, first service pregnancy per AI did not differ (PGF+AIE = 42.0%; PGF+TAI = 47.3%, ALL-TAI = 43.8%). Time to pregnancy was reduced for ALL-TAI compared with PGF+AIE (HR = 1.20, 95% CI = 1.02 to 1.42), but was similar to that of PGF+TAI (HR = 1.13, 95% CI = 0.96 to 1.33). Time to pregnancy did not differ for PGF+AIE and PGF+TAI (HR = 1.07, 95% CI = 0.91 to 1.25). Median days to pregnancy were 27, 23, and 21 for heifers in PGF+AIE, PGF+TAI, and ALL-TAI, respectively. We concluded that an ALL-TAI program for first service reduced time to pregnancy, albeit a relatively small reduction, when compared with a program that relied primarily on AIE after induction of estrus with PGF2α treatments. The program that combined synchronization of estrus and TAI (PGF+TAI) resulted in similar time to pregnancy than the predominant TAI and predominant AIE programs.

Revisiting the impact of ejaculatory abstinence on semen quality and intracytoplasmic sperm injection outcomes.

Regulatory bodies recommend inconsistent ejaculatory abstinence lengths before semen analysis. The literature exploring the effect of ejaculatory abstinence length on the outcomes of intracytoplasmic sperm injection is scarce. To study the influence of ejaculatory abstinence length on semen quality and intracytoplasmic sperm injection outcomes. This prospective cohort study included 818 patients undergoing conventional semen analysis from October 2015 to October 2016, in a private university-affiliated IVF centre. Generalized linear models adjusted for potential confounders were used to investigate the associations between ejaculatory abstinence length and seminal parameters and intracytoplasmic sperm injection outcomes. Increasing ejaculatory abstinence length was positively correlated with semen volume, sperm concentration, total sperm count, total motile sperm count and sperm DNA fragmentation index. Significant inverse correlations were observed between ejaculatory abstinence length and fertilization rate, blastocyst formation rate, implantation rate and pregnancy rate. A discriminant analysis showed a mean ejaculatory abstinence length in the positive pregnancy group of 3.14±1.64days and 4.83±3.66days in the negative pregnancy group. A cut-off point was established halfway between ejaculatory abstinence length averages, at 4days. The ejaculatory abstinence ≤4days group showed significant lower semen volume, sperm concentration, total sperm count and total motile sperm count compared to ejaculatory abstinence>4days group. The ejaculatory abstinence≤4days group showed significant lower sperm DNA fragmentation index, and higher rates of fertilization, high-quality embryos on day 3, blastocyst development, implantation and pregnancy compared to ejaculatory abstinence>4days group. The implantation rate was significantly higher and the pregnancy rate tended to be higher with one day of ejaculatory abstinence, compared to 2-4days of ejaculatory abstinence. Ejaculatory abstinence periods of >4days have a detrimental effect on sperm DNA and intracytoplasmic sperm injection outcomes. One day of ejaculatory abstinence significantly improves implantation rate and tends to increase pregnancy rate, compared to 2, 3 and 4days of ejaculatory abstinence.

Sperm solute carrier family 9 regulator 1 is correlated with boar fertility

Predicting male fertility is extremely important for artificial insemination and profitable farm management. Conventional semen assessment together with computer-assisted sperm analysis is widely used to predict male fertility under field conditions. However, the clinical validation and sensitivity of these methods remain unclear. Therefore, a new approach is needed to predict male fertility. Here, we investigated the use of a transcriptomic marker (solute carrier family 9, subfamily A, member 3, regulator 1; SLC9A3R1) together with sperm motility parameters and capacitation status to predict fertility/infertility in boars at the commercial level. Our data showed that among motility parameters and the capacitation status, hyperactivation (HYP) differed between high- and low-litter size boars. HYP showed a significant positive correlation (R = 0.468) with boar litter size. Simultaneously, the expression of SLC9A3R1, a gene important in sperm ion channel regulation, was significantly negatively correlated (R = −0.523) with boar litter size. Quality assessment revealed that both HYP and SLC9A3R1 showed considerable sensitivity (71.43 vs. 100%), specificity (100 vs. 71.43%), and overall accuracy (90%) for predicting male fertility. Interestingly, the potential of SLC9A3R1 expression to increase the average piglet number per breeding was higher (0.7 piglets) than that of HYP (0.5 piglets). Thus, measuring SLC9A3R1 expression in spermatozoa may be a more accurate marker for evaluating male fertility/infertility than conventionally used motility parameters and capacitation status.

Seasonal variations in hormones and enzymes of seminal plasma and its relationship with semen quality in crossbred cattle bulls

ABSTRACTPresent study was designed to assess the seasonal influence of seminal plasma constituents on sperm attributes in crossbred cattle bulls. Semen ejaculates were collected from 10 crossbred Karan Fries bulls at fortnightly interval during hot-humid and winter months. The seminal plasma composition (hormones and enzymes) was evaluated using standard kits. ALT, AST and ALP activities were higher during hot-humid months. Seasonal variations were not observed in testosterone and prostaglandin E2, whereas estradiol showed a significantly higher level during hot-humid season. Moreover, testosterone and estradiol showed no correlation with sperm characteristics. Prostaglandin E2 was negatively correlated (p < 0.05) with sperm abnormality. The enzyme levels were found to be related to semen quality attributes. AST showed significant negative correlation with mass activity, individual motility, sperm viability and a positive correlation (p < 0.01) with the abnormal sperms. ALT activity negatively correlated to mass activity (p < 0.05). A negative correlation of ALP with mass activity, sperm viability and sperm membrane integrity and positive correlation with abnormal sperm percent were also observed. It was concluded that seminal plasma enzymatic activity if coupled with conventional semen analysis parameters was better indicator of semen quality. Presence of reproductive hormones in seminal plasma did not influence the semen quality after ejaculation.

7 Pregnancy rates in suckled beef cows synchronized with a shortened progesterone/oestradiol-based protocol (J-synch) and inseminated with conventional or sexed-sorted semen

An experiment was designed to evaluate pregnancy rates (P/AI) in suckled beef cows synchronized with a progesterone (P4)/oestradiol-based protocol (J-Synch) and inseminated (AI) with sexed or nonsexed (conventional) semen. Angus or Angus crossbred cows (n=877), 60 to 90 days postpartum, with a body condition score of 2.5 to 3.5 (scale of 1 to 5) and a corpus luteum or a follicle &amp;gt;8mm in diameter detected by ultrasonography, were randomly allocated to be AI with sexed-sorted or nonsexed (conventional) semen. The experiment was performed in 4 farms, and the sexed-sorted (SexedUltra™ 4M, with 4 million sperm; ST Genetics, Navasota, TX, USA) and conventional (25 million sperm) semen were from split ejaculates from 5 Angus bulls. All cows received a P4 device (DIB 0.5, Zoetis, Villa Adelina, Argentina) and 2mg of oestradiol benzoate (Gonadiol, Zoetis) on Day 0. On Day 6, cows received 400IU of eCG (Novormon 5000, Zoetis) and 500µg of cloprostenol (Ciclase DL, Zoetis) and DIB was removed. All cows were also tail painted and observed for signs of oestrus (i.e. &amp;gt;30% of the tail paint rubbed off) at 60, 72, and 84h after DIB removal. Cows with the tail paint rubbed off by 60 or 72h after DIB removal were artificially inseminated at 72h with either sexed or conventional semen. Those not showing oestrus by 72h received 100µg of gonadorelin acetate (Gonasyn GDR, Zoetis) at that time and were artificially inseminated with either sexed-sorted or conventional semen 12h later (i.e. 84h). Pregnancy was diagnosed by ultrasonography 30 days after AI. Data were analysed by general linear model for binary data with a logit link. Overall, 76.4% (678/877) of cows showed oestrus 60 to 84h after DIB removal, and there were differences in P/AI between sexed-sorted and conventional semen (P&amp;lt;0.01; Table 1); however, there was no farm effect (range: 52-59%) and no interactions. Fertility differed (P&amp;lt;0.05) among bulls and ranged from 25.8 to 55.3% for sexed-sorted semen and from 53.1 to 73.9% for conventional semen. In cows artificially inseminated with sexed-sorted semen, P/AI was the highest in cows showing oestrus and AI at 84 h; however, no differences were detected among cows artificially inseminated with conventional semen (Table 1). Although P/AI was lower for sexed-sorted than conventional semen, tail painting for oestrus detection can be successfully applied to AI beef cows with sexed-sorted semen synchronized with a shortened P4/oestradiol-based protocol. Table 1.Effect of semen type (sexed-sorted or conventional) and time of oestrus and AI on P/AI in suckled beef cows synchronized with a shortened P4/oestradiol-based protocol Research was supported by Fondo Nacional de Ciencia y Tecnología (FONCYT PICT 2017-4550) and UNVM.

140 A comparison of in vitro embryo production between heifers and lactating Holstein donors without superstimulation

Bovine oocyte donor age seems to have an important role on in vitro embryo production. Therefore, the aim of this study was to compare cumulus-oocyte complex (COC) recovery, cleavage, and blastocyst rates between heifers and lactating Holstein donors. A total of 89 animals (heifers: n=60, 11 to 17 months of age; lactating cows: n=29, 60 to 180 days in milk) were used in this experiment. Ovum pickup (Day −1) was performed on a random day of the oestrous cycle without superstimulation by a single technician. Conventional or sexed semen were used for IVF (Day 0). Cleavage and blastocyst rates were evaluated on Days 3 and 7, respectively. Cleavage was determined when structures had 4 or more cells and blastocyst rate included grades I and II blastocysts. Continuous data were analysed by t-test, and binomial data were analysed by Fischer’s exact test. A total of 1,289 COC were recovered from lactating cows (n=509) and heifers (n=777). Total and viable COC per donor were higher (P&amp;lt;0.05) in lactating cows (total=17.55±13.08; viable=11.24±8.9) than heifers (total=12.95±5.39; viable=7.88±4.08). The percentage of viable COC was similar (P&amp;gt;0.05) in lactating cows (64.04%; 326/509) and heifers (60.87%; 473/777). Cleavage rate was higher (P&amp;lt;0.0001) in lactating cows (58.74%; 299/509) than in heifers (45.05%; 350/427). Cleavage rate was similar (P&amp;gt;0.05) with conventional and sexed semen in heifers (conventional=37.68%; 52/138; sexed=46.63%; 298/639) and cows (conventional=55.05%; 49/89; sexed=59.52%; 250/470). Blastocyst rate was higher (P&amp;lt;0.0001) in lactating cows (19.84%; 101/509) than in heifers (8.49%; 66/777). Conventional semen had a higher (P&amp;lt;0.0001) blastocyst rate (18.11%; 24/138) than sexed semen (6.57%; 42/639) in heifers. However, there was no difference (P&amp;gt;0.05) in blastocyst rate between conventional (24.71%; 22/89) and sexed semen (18.8%; 79/420) in lactating cows. Blastocyst/cleaved structures ratio was higher (P&amp;lt;0.05) in heifers using conventional (46.15%; 24/52) compared with sexed (14.06%; 42/298) semen. However, in lactating cows there was a tendency (P&amp;lt;0.1) for higher blastocyst/cleaved structures ratio using conventional (44.89%; 22/49) compared with sexed semen (31.6%; 79/250). In conclusion, a higher number of total and viable COC were aspirated from Holstein lactating cows than heifers. Furthermore, cleavage and blastocyst rates were higher in lactating cows than in heifers. Conventional semen gave more embryos than did sexed semen in heifers but not in cows. Overall, oocytes from Holstein lactating donors are more suitable for in vitro embryo production than are those from heifer donors.

130 In vitro embryo production using frozen semen from cloned and non-cloned Bos indicus bulls

Reproductive biotechnology has continued to evolve rapidly, allowing the development of techniques to increase reproductive efficiency and contribute to the genetic improvement of cattle. Some of these techniques include the in vitro maturation and IVF of oocytes, sperm sexing and cloning. These modern assisted reproductive techniques can help produce offspring of desired genetic characteristics and of a pre-determined sex. However, studies of the bull’s contribution to in vitro reproductive performance are scarce in the Brahman breed. The aim of this study was to compare oocyte maturation and embryo production in vitro using frozen semen from 5 Brahman bulls (Bos indicus), cloned (n=1) and non-cloned (n=4), with characteristics and genetics of high commercial value. The age of the bulls at the time of semen collection and cryopreservation ranged from 2 to 7 years. The oocytes were obtained on 2 different dates (45 days between collections) using pooled oocytes collected by ovum pickup at random stages of the oestrous cycle, from a total of 15 Brahman donor cows. Oocytes were transported to a laboratory in the State of Chiapas, Mexico (Genemex Internacional). The oocytes were cultured in maturation medium for 24h. For IVF, conventional semen was used from one bull (B1) and his clone (B12), the grandson of B1 (B2), and 2 non-related bulls (B3 and B4). The gametes were co-incubated for 22h and afterward placed in medium for embryo development and cultured for 7 days in a humid atmosphere with 5% CO2 in air. Of the matured oocytes, 36/43 (84%), 16/32 (50%), 101/143 (70%), 46/67 (68%) and 53/65 (81%) were fertilized using semen from B1, B12, B2, B3 and B4, respectively. Of the fertilized oocytes, 15/30 (50%), 8/16 (50%), 45/101 (44%), 21/46 (45%) and 18/53 (34%) resulted in transferrable embryos, corresponding to semen from the same bulls, respectively. This would appear to be the first scientific report in Mexico about the use of semen from a cloned bull for in vitro embryo production. In IVF, similar results were observed between B1 and a non-related bull (B4). Similar results in transferrable embryos were observed between B1 and B12 but also similar to a related bull (B3) and a non-related bull (B4). A Fisher’s exact test of the IVF results comparing B1 and B12 found a significantly (P&amp;lt;0.05) higher number of fertilized oocytes for B1. However, a significant difference was not found (P&amp;gt;0.05) concerning the number of transferrable embryos produced by these two bulls. In conclusion, the Brahman bulls in this study differ in their contribution to IVF and embryo production. Further studies are required to determine the factors responsible for such effects, e.g. age differences or clone versus non-clone mosaicism. Results from this research contribute to the study and development of assisted reproductive techniques for increasing in vitro production efficiency in Zebu cattle. We thank the Rosales family, from El Herradero Ranch, in the State of Campeche, Mexico, for allowing the use of their cattle for this project.

87 Assessment of pregnancy success following transfer of embryos produced in vitro using frozen - thawed semen from cloned and noncloned Bos indicus bulls

Assisted reproductive technologies (ART) continue to develop rapidly, allowing for the development of techniques to increase reproductive efficiency and contribute to the genetic improvement of cattle. Some of these techniques include in vitro production (IVP) of embryos and embryo transfer. These modern ART can help produce offspring with highly desirable characteristics. However, there is a lack of information on the percentage of pregnancies obtained following transfer (P/ET) of IVP embryos derived using semen of cloned Bos indicus bulls. The objective of this study was to compare embryo transfer results of IVP embryos created using frozen-thawed semen from 5 Brahman bulls (Bos indicus) with characteristics and genetics of high commercial value. The embryos were produced on two different dates, 45 days apart, using pooled oocytes harvested by ovum pickup from 15 Brahman cows at random stages of the oestrous cycle. Procedures for IVP were performed in a commercial laboratory (Genemex Internacional) in the state of Chiapas, Mexico. For IVF, conventional semen was used from 1 bull (B1) and his clone (B12), the grandson of B1 (B2), and from 2 nonrelated bulls (B3 and B4). A total of 100 embryos were transferred nonsurgically by a private practitioner on a ranch in the state of Campeche, Mexico. The recipients were commercial crossbred cows synchronized using a FTET program. On Day 0, recipients received an intravaginal device containing 1.9g of progesterone (CIDR) and 2mg of oestradiol benzoate IM. On Day 8, the CIDR was removed and cows received 25mg of dinoprost tromethamine, 200IU of eCG, and 0.5mg of oestradiol cypionate IM. Embryos were transferred on Day 17. The overall P/ET was 42% (42/100). The P/ET for IVP embryos produced with semen from bulls B1, B12, B2, B3, and B4 was 3/15 (20%), 3/8 (37%), 23/42 (55%), 8/20 (40%), and 5/15 (33%), respectively. The P/ET was numerically greater for embryos produced using semen from the cloned bull (37%; B12) compared to embryos produced using semen from the original noncloned bull (20%; B1), although this difference was not statistically significant (P=0.62, Fisher’s exact test). There was a significant difference (P&amp;lt;0.05) for the P/ET obtained with embryos produced using semen from bulls B1 and B2, but results for the other bulls were not significantly different. As far as we know, this is the first scientific report in Mexico concerning the use and comparison of semen from cloned and noncloned bulls for the production and transfer of bovine IVP embryos. In general, a wide numerical range of P/ET using the different bulls was observed (i.e. 20-55%). In this preliminary study, there was no impact of using frozen-thawed semen from a cloned bull for IVP on P/ET. The results from this research can contribute to the study and development of ART to improve P/ET obtained using Zebu IVP embryos. However, further research with a larger numbers of animals is required to confirm whether using semen from cloned and noncloned Bos indicus bulls for IVP impacts pregnancy success following embryo transfer.

A simulation model to evaluate the economic consequences of insemination programs in dairy herds: timed artificial insemination and sex-sorted semen

ABSTRACT The objective of this study was to develop a simulation model to analyse the technical, economic, and financial performance of using different reproductive strategies in dairy herds. Strategies simulated were: artificial insemination (AI) using conventional semen after oestrus detection (AIC), AI using sex-sorted semen after oestrus detection (AIS), timed artificial insemination (TAI) using conventional semen (TAIC), and TAI using sex-sorted semen (TAIS). The total time horizon analysed corresponded to 25 years, divided into 425 periods of 21 days. The [...]

Mitochondrial functionality modifies human sperm acrosin activity, acrosome reaction capability and chromatin integrity.

In addition to sperm motility, which major biological characteristics of sperm fertility potential are associated with mitochondrial functionality? Sperm fertilization capacities, including acrosin activity, acrosome reaction (AR) capability and chromatin integrity, are related to the mitochondria functionality as evaluated by the mitochondrial membrane potential (MMP). Correlative studies suggest a potential role of sperm MMP in predicting sperm fertilization ability and ensuring sperm motility. However, researches characterizing other determinants of sperm fertility potential according to MMP are lacking. The sperm MMP was examined in 627 young college students in the Male Reproductive Health in Chongqing College Students (MARHCS) cohort study in 2014. Among these participants, acrosin activity and chromatin integrity were measured in 378 and 604 subjects, respectively. These two determinants of sperm fertility potential were first compared among high-, moderate- and low-MMP groups in the college population. The effects of MMP collapse caused by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on acrosin activity, AR, DNA fragmentation, reactive oxygen species (ROS) production, and ATP content in human spermatozoa were evaluated in vitro. The sperm MMP was evaluated by using JC-1 staining, acrosin activity was measured using a N-α-benzoyl-dl-arginine-para-nitroanilide HCl (BAPNA) substrate method, the integrity of chromatin represented by DNA fragmentation index (DFI) was measured by sperm chromatin structure assay (SCSA), AR was evaluated with chlortetracycline staining, and intracellular ROS production was evaluated with dihydroethidium. ATP concentration was determined with luciferase. Measurements were performed by spectrophotometry or flow cytometry. Nonparametric analysis revealed significantly higher acrosin activity and a lower DFI in subjects with moderate or high MMP compared to those with low MMP. After adjustment for potential confounders, increases of 7.9 and 44.4% in sperm acrosin activity and deceases of 12.0 and 25.2% in the sperm DFI were found in the moderate- and high-MMP groups, respectively. The MMP dissipation induced by CCCP caused significant declines in acrosin activity and AR capacity and increased DFI in human spermatozoa. Moreover, sperm MMP dissipation induced ROS overproduction and decreased ATP content. We cannot exclude a contribution of leukocytes to ROS production and no size gating was used to exclude these cells from the FACS measurements. No simultaneous live-dead staining was done and a contribution of dead sperm to the MMP and acrosome assays cannot be excluded. Mitochondrial functionality might be necessary to maintain sperm acrosin activity, AR and chromatin integrity. Tests of mitochondrial functionality should be developed and used independently of or in addition to conventional semen parameters in infertility diagnosis or risk-assessment processes. This study was supported by the Key Program of the National Natural Science Foundation of China (No. 81630087) and the National Natural Science Foundation of China (No. 81703254). None of the authors have any competing interests to declare.

Fertility of lactating dairy cows inseminated with sex‐sorted or conventional semen after Ovsynch, Presynch–Ovsynch and Double‐Ovsynch protocols

The objective was to compare pregnancy per artificial insemination (P/AI) with conventional (CS) or sex-sorted semen (SS) in dairy cows subjected to one of the three timed AI protocols. Cows (n=356) were randomly assigned to synchronization with Ovsynch (OVS), Presynch-Ovsynch (PO) or Double-Ovsynch (DO) and inseminated on Day 77±3 postpartum with either frozen-thawed SS (n=182) or CS (n=184) of the same bull. More cows were cyclic at the beginning of breeding Ovsynch increased (p<0.01) with presynchronization and it was greater for DO than PO (OVS=78.5%, PO=85.1%, DO=95.6%). Overall, P/AI for SS and CS increased with presynchronization (p<0.05) on Days 31 (OVS=35.5%, PO=47.1%, DO=48.3%) and 62 (OVS=30.1%, PO=43.8%, DO=43.9%). Regardless of synchronization treatments, insemination with SS reduced P/AI (p<0.02) on Days 31 (38.1% vs. 50.6%) and 62 (34.5% vs. 45.6%) compared with CS. No interaction was observed between synchronization treatment and type of semen for P/AI, although in cows receiving CS, P/AI was numerically greatest for PO (OVS=42.0%, PO=59.3%, DO=49.0%), and in cows receiving SS, it was numerically greatest for those inseminated following DO (OVS=27.9%, PO=35.5%, DO=47.6%). Thus, presynchronization improved P/AI in cows inseminated with sex-sorted or conventional semen.