scholarly journals Evaluation of in vitro production rates of bovine embryos using melatonin-supplemented culture medium

2021 ◽  
Vol 10 (6) ◽  
pp. e19010615544
Author(s):  
Ricardo Magalhães ◽  
Carlos Renato de Freitas Guaitolini ◽  
Marcio Luiz Denck Tramontin ◽  
Danielle Andressa Oliveira Sestari ◽  
Bruno Argenton de Barros ◽  
...  
Keyword(s):  
Culture Medium ◽  
Statistical Significance ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
In Vitro Production ◽  
Embryo Production ◽  
Vitro Fertilization

In this study, we aimed to evaluate the rate of bovine embryo production by using 50 ng/mL melatonin supplementation in in vitro culture medium. For this, oocytes from slaughterhouse ovaries were matured in vitro in TCM-199 medium with Earle’s balanced salt solution + 10% SFB, FSH, and LH in an atmosphere of 5% CO2. Twenty-four hours after IVM, the oocytes underwent in vitro fertilization in human tubal fluid under the same conditions as above, for 18 h. Semen was fractionated by Percoll gradient centrifugation and the concentration of sperm was adjusted to 1 × 106/mL. Probable zygotes were then divided into two groups: the control group grown in drops of 90 μL SOFaa medium + 0.6% BSA + 2.5% SFB, in an atmosphere of 5% CO2, 90% N2, and a melatonin group (Mel), similarly cultured in 90 μL drops of SOFaa medium + 0.6% BSA + 2.5% SFB + 50 ng/mL melatonin. Cleavage rates were assessed on day 3 (D3). On D7, blastocyst formation rates were evaluated. Eight routines were performed (320 oocytes per routine). Data were analyzed with ANOVA, followed by Tukey’s range test using a general linear model. The level of statistical significance was set at 5%. There were no differences in the rates of cleavage or blastocyst formation between the control and melatonin groups (P > 0.05). Thus, under the conditions used in this study, supplementation with melatonin did not yield benefits in increasing the rate of in vitro bovine embryo production.

scholarly journals The role of Kisspeptin in bovine in vitro embryo production

2018 ◽  
Vol 39 (2) ◽  
pp. 621 ◽  
Author(s):  
Mayara Mafra Soares ◽  
Deize De Cássia Antonino ◽  
Mayara Oliveira ◽  
Jairo Melo Júnior ◽  
Luciana Ribeiro Peixoto ◽  
...  
Keyword(s):  
Control Group ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
The Third ◽  
Bovine Reproduction ◽  
Vitro Production

The aim of this study was to investigate the effect of Kisspeptin (Kp) on the medium used in different stages of in vitro production of bovine embryo (IVEP), evaluating cleavage (CR) and blastocyst (BR) rates. The study was divided into three experiments that analyzed, respectively, the action of Kp on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of bovine embryos. In experiment 1, the oocytes were matured in IVM medium and distributed into the following treatments: maturation (IVM Control, n = 102), maturation with addition of 10-7 M Kp (Kp 10-7 IVM, n = 90), and hormone-free maturation luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with the addition of 10-7M Kp (No hormones + Kp 10-7, n = 84), following maturation to normal stages of IVEP. In experiment 2, the oocytes were fertilized in IVF medium, in the following treatments: TALP-FERT without Kp (Control IVF, n = 103) and TALP-FERT with the addition of 10-7M Kp (Kp 10-7 IVF, n = 119), usually following the other steps. Finally, in the third experiment, the oocytes passed through all phases and were divided into IVC in two treatments: SOF medium without Kp (Control IVC, n = 109) and SOF medium with the addition of 10-7M Kp (Kp 10-7, N = 106). The data were analyzed by PROC GLIMMIX of the SAS program. In experiment 1, the means of CR and BR were similar (P > 0.05) between treatments (IVM Control76.47% and 37.25%, Kp 10-7 MIV80% and 33.33%, and No hormones + Kp 10-770.24% and 30.95%, respectively). In experiment 2, the means of CR were similar for the IVF Control and Kp 10-7 IVF groups (P > 0.05), 76.70% and 86.55% respectively. But, the mean of the BR of the group Kp 10-7 IVF was 38.66%, which was higher (P < 0.05) than that of the FIV Control group, which was 31.07%. In the third experiment, the means of CR and BR (P > 0.05) were similar between the IVC Control and Kp 10-7 IVC groups (CR 83.50% and 78.30%, and BR 26.60% and 23.60%, respectively). Although at this concentration of 10-7M during IVC no change in embryo production is seen, Kp presents the same performance as both gonadotrophins in oocyte maturation and modulates the fertilization process, providing more blastocysts. With these findings, it can be seen that Kp presents a regulatory action on bovine reproduction, and can be an excellent tool to maximize IVEP indexes.

Male-dependent variability of fertilization and embryo development in two bovine in vitro fertilization systems and the effects of casein phosphopeptides (CPPs)

1997 ◽  
Vol 9 (4) ◽  
pp. 465 ◽  
Author(s):  
U. Kreysing ◽  
T. Nagai ◽  
H. Niemann
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Individual Variability ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Casein Phosphopeptides ◽  
Fertilization System

This study investigated the effects of semen from five different bulls and two different ejaculates of the same bull on penetration, cleavage, blastocyst formation, and cell allocation in bovine blastocysts produced in vitro. Casein phosphopeptides (CPPs) were tested for their ability to enhance fertilization and minimize variability among bulls and ejaculates. In Experiment 1, the BO-fertilization system was employed. Penetration and polyspermy both displayed great variation among bulls and between ejaculates, whereas no significant differences were observed in cleavage and blastocyst-formation rates. Similar variability was observed in penetration, polyspermy, cleavage, blastocyst-formation rates and cell allocation and distribution when the two fertilization systems, TALP and BO, were compared in Experiment 2. The BO-system supported penetration and polyspermy better (P < 0·05) than the TALP-system, whereas the TALP-system was superior (P < 0·05) in supporting cleavage and blastocyst formation. Significant interactions existed between bulls and the fertilization system employed. It is concluded that the success of in vitrofertilization is markedly dependent on individual bulls as well as on ejaculates from the same bull. CPPs are able to enhance penetration and embryo development in certain bulls or ejaculates and thus contribute to reducing the degree of individual variability, but they do not generally improve the success of bovine embryo production in vitro.

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

scholarly journals Modern achievements of the development of scientific research at obtaining in vitro sheep embryos

2020 ◽  
Vol 22 (93) ◽  
pp. 60-68
Author(s):  
O. M. Sharan ◽  
V. Yu. Stefanyk ◽  
S. G. Shalovylo
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Culture ◽  
Biologically Active ◽  
In Vitro Production ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Maturation In Vitro ◽  
Key Factor ◽  
Vitro Production

New literature data on research aimed at improving the in vitro production of sheep embryos presents in the article. An analysis of the achievements of scientists from different countries to increase the efficiency of the main stages of embryo production in vitro: maturation of oocytes in vitro, their in vitro fertilization and in vitro embryo culture. In the literature experience has shown that the efficiency of oocyte maturation in vitro is significantly influenced by the experience and qualifications of scientists, the age of the egg donor, the improvement of the environment by adding roscovitin to inhibit meiosis, α-linolenic acid, cerium dioxide nanoparticles (CeO2 NPs) and sericin to accelerate nuclear maturation and increase the number of oocytes of the second meiotic metaphase (MII). The main factors influencing the effectiveness of in vitro fertilization have been identified, and the parameters of the limited time of fertilization ability of sperm and the ability of oocytes to fertilize, which is called the “fertile span”, have been determined. The main effective medium that increases the effectiveness of in vitro fertilization – synthetic oviduct fluid (SOF) with the addition of heparin and serum of cattle or sheep. The main parameters of sheep embryo culture in vitro are presented with the definition of the most commonly used media and their influence on embryonic development. Potential ways to improve the production of sheep embryos in vitro with the determination of morphological evaluation of categories of oocytes, methods of synchronization of their maturation in vitro are also highlighted. At the same time, literature data on the synchronization of oocyte-cumulus complexes with the use of a large number of inhibitors of meiotic division are presented, which according to many scientists may be a key factor in improving the efficiency of sheep embryo production in vitro. In addition, the results of studies of many scientists on the expansion of the fertile gap of oocytes of sheep cultured in vitro using certain biologically active substances were analyzed. In conclusion, the prospect of using the technology of in vitro production of sheep embryos in biomedical research is highlighted.

186 THE EFFECT OF MATRIGEL ON THE DEVELOPMENT OF IN VITRO-FERTILIZED PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 209
Author(s):  
S.-W. Kim ◽  
M.-J. Lee ◽  
B.-C. Yang ◽  
G.-S. Im ◽  
H.-H. Seong ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Statistical Significance ◽  
Control Group ◽  
Fertilization In Vitro ◽  
Mouse Tumor ◽  
Matrix Product ◽  
Vitro Fertilization ◽  
Culture Systems

The application of matrix proteins to culture systems for growth of embryos is a logical extension in the quest to better simulate the in vivo culture environment. Matrigel, a commercially available extracellular matrix product containing collagen IV, laminin, entactin, and proteoglycans isolated from mouse tumor cells, has been tested. Development of mouse pre-implantation embryos cultivated in conventional culture medium was contrasted to that of embryos grown in solubilized Matrigel medium. In the solubilized Matrigel medium, in vitro blastocyst formation and hatching were significantly enhanced over that observed in the medium alone control. Therefore, the aim of this study was to investigate the effect of solubilized Matrigel on the development of porcine embryos after in vitro fertilization. In vitro-matured oocytes were fertilized in mTBM medium with fresh spermatozoa for 6 h. Putative zygotes were cultured in PZM-3 medium supplemented with (matrigel group) or without (control group) 0.8% Matrigel for 6 days. The number of cells in blastocysts was determined by staining with Hoechst 33342. Assessment of apoptosis in blastocysts was examined by TUNEL. The statistical significance of the data was analyzed using chi-square test and Student&apos;s t-test. The addition of Matrigel appeared not to increase the proportion of blastocysts (control: 71/219, 21.8 � 2.2% vs. Matrigel: 69/220, 23.5 � 5.8%). However, the mean cell numbers were significantly increased by Matrigel (Matrigel: n = 31, 52.9 � 18.1 vs. control: n = 30, 42.3 � 14.4; P &lt; 0.01). The proportion of apoptotic cells was significantly lower in the Matrigel group (Matrigel: 4.5 � 4.2% vs. control: 6.6 � 5.5%; P &lt; 0.05). In this experiment, Matrigel appeared to increase blastocyst quality of porcine embryos. Results suggest that Matrigel, as an extracellular matrix component, may be another avenue for formulating more physiological culture systems.

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 250
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
V. P. Oliveira ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Electric Pulses ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
And Control

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P < 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 205 ◽  
Author(s):  
E. Mullaart ◽  
F. Dotinga ◽  
C. Ponsart ◽  
H. Knijn ◽  
J. Schouten
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Calf Serum ◽  
High Serum ◽  
In Vitro Production ◽  
Embryo Production ◽  
Chi Square ◽  
Embryo Yield ◽  
Vitro Production

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated. Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum

238 KNOCKING DOWN OF THYROID HORMONE RECEPTORS INHIBITS DEVELOPMENT OF EARLY BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
N. Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Reference Genes ◽  
Messenger Rna ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Mrna Transcripts ◽  
In Vitro Embryo Production

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated. Funding was provided by CRC, NSERC, and the EmbryoGENE network.

7 THE EFFECT OF A NOVEL RECOMBINANT PROTEASE TREATMENT ON BOVINE SPERM FERTILIZING CAPACITY AND EMBRYO DEVELOPMENT IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 104
Author(s):  
J. T. Aaltonen ◽  
K. J. Mattson ◽  
N. M. Loskutoff
Keyword(s):  
Embryo Development ◽  
Disease Transmission ◽  
Bos Taurus ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Bovine Embryo ◽  
Human Sequence ◽  
Bovine Sperm

As described in the IETS Manual (Stringfellow and Seidel, 1995), and endorsed by the OIE, trypsin can be used (for specific pathogens and livestock) to effectively remove certain infectious agents from in vivo-derived embryos for international transport. Because of the multimillion-dollar AI industry for livestock, the OIE has encouraged more research in developing similar decontamination techniques for semen as an added safeguard to animal quarantine for the prevention of disease transmission. Most or all of the earlier studies on embryos used a porcine pancreatic-derived trypsin. Because of more stringent guidelines from international regulatory agencies on the use of animal products, several serine protease recombinants are now available. Previous experiments comparing the porcine pancreatic extract with a recombinant bovine sequence trypsin developed in corn resulted in no statistical difference in cleavage or morula/blastocyst rates. (Mattson et al. 2008 Theriogenology 69, 724–727). An additional in vivo study treating bovine sperm with a yeast-derived human-sequence trypsin resulted in significantly more transferable-quality embryos after the AI of superovulated cows as compared with sperm not treated with trypsin (Blevins et al. 2008 Reprod. Fertil. Dev. 20, 84). The goal of this experiment was to examine the in vitro development of bovine embryos produced from sperm treated with a recombinant trypsin found in a commercially available density gradient centrifugation (DGC) product (Bovipure, Nidacon, Sweden) compared with DGC without trypsin. Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 5 replications (n = 2220 oocytes). Semen was collected and pooled from 2 Bos taurus bulls and frozen in an egg-yolk cryodiluent (Biladyl, Minitube). The semen was processed using Bovipure DGC composed of 2 mL of 40% colloid of silane-coated silica particles containing either a yeast-derived human sequence recombinant trypsin containing no animal by-products (n = 1126 oocytes) or the same colloid without trypsin as the control group (n = 1094 oocytes). Both 40% concentrations were layered over 2 mL of an 80% concentration of the same colloid without any additives. The density gradients were centrifuged at 300g for 20 min, after which time the pellets were washed in 5 mL of prewarmed TL Hepes solution (Cambrex) and centrifuged at 500g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 106 sperm mL–1, to be used for in vitro inseminations. Results were compared using a 2-tailed unpaired t-test. Cleavage rates for the trypsin-treated sperm (n = 969, 35.8%) and the control (n = 950, 44.3%) groups were not statistically different (P = 0.20). Although more embryos reached the morula to blastocyst stages in the control group (n = 421, 61.0%) than in the trypsinized group (n = 347, 54.7%), these differences also were not statistically significant (P = 0.85). In conclusion, trypsinized Bovipure DGC of sperm before insemination showed no detrimental effects on IVF-derived bovine embryo development.

Sperm recovery techniques to maximize fertilizing capacity

1994 ◽  
Vol 6 (1) ◽  
pp. 25 ◽  
Author(s):  
D Mortimer
Keyword(s):  
Culture Medium ◽  
Lower Layer ◽  
Gradient Centrifugation ◽  
Vitro Fertilization ◽  
Recovery Techniques ◽  
Pharmacological Stimulation ◽  
Percoll Gradients ◽  
Sperm Dysfunction ◽  
Selection Of

Because seminal plasma contains factors that inhibit the fertilizing ability of spermatozoa, it is essential that spermatozoa be separated from it quickly and efficiently. Although the success of a sperm preparation method is often assessed by the yield of motile spermatozoa, the choice of a method also depends on its technical complexity, the materials and apparatus required and time costs. Any exposure of spermatozoa during preparation to factors that may cause iatrogenic sperm dysfunction must obviously be avoided. Consequently, methods involving centrifugal washing prior to the selection of motile spermatozoa should be avoided. Direct swim-up from semen is the simplest way to obtain highly motile sperm populations and can be a very rapid procedure with normal semen samples. Two-layer discontinuous Percoll gradients give excellent yields when the lower layer contains 81% (v/v) Percoll. However, for severely asthenozoospermic cases the results can be disappointing and a Nycodenz gradient may be better, although the 'mini-Percoll' technique might be useful if special care is taken to protect the spermatozoa from damage induced by free radicals. In such cases the migration-sedimentation approach can also be successful. Abnormal samples, especially those with increased viscosity, may benefit from prior dilution with culture medium, or even chymotrypsin-induced liquefaction, before density gradient centrifugation. Finally, pharmacological stimulation of sperm motility may increase yields but, for in vitro fertilization (IVF), such spermatozoa must be used to inseminate oocytes as soon as possible after exposure to the stimulant, although after its removal.

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