Inseminating dose for the artificial fertilization of Brycon amazonicus (Teleostei: Characidae)

Zygote ◽  
2021 ◽  
pp. 1-4
Author(s):  
Rosilane Gomes de Souza de Oliveira ◽  
Marle Angélica Villacorta-Correa

Summary Knowledge of the sperm–oocyte ratio in fish fertilization serves as the basis for studies on artificial reproduction and gamete manipulation. The aim of this study was to determine the minimum insemination dose for Brycon amazonicus oocyte fertilization. Female and male gametes were used and tested with the following doses of spermatozoa oocyte–1 ml–1: 10,000, 20,000, 40,000, 60,000 and 80,000 (in triplicate). Fertilization rates were calculated and estimated from the regression equation by applying the segmented regression model ‘Linear Response Plateau’ to determine the appropriate proportion of gametes. Based on the equation Ŷ = 14.3415 + 0.0007836X, the fertilization rate increased up to 63.34% as it reached a plateau with a proportion of 62,524 spermatozoa oocyte–1 ml–1, which is the minimum insemination dose recommended for artificial insemination of the species.

2005 ◽  
Vol 17 (2) ◽  
pp. 155
Author(s):  
F. Becker ◽  
H. Alm ◽  
F. Schneider ◽  
H. Nehring ◽  
L. Rothe ◽  
...  

Estrus detection and determination of time of insemination are very important factors in reproduction management of cattle. Therefore an estrus synchronization schedule in combination with induction of ovulation and a single insemination at a predetermined time in dairy cattle was established to achieve high pregnancy rates (Kanitz etal. 2002 Reprod. Nutr. Dev. 42, 587–599; Becker et al. 2004). The aim of the recent study was to investigate the influence of the number of spermatozoa per insemination dosage on embryo development and the interrelationship between number of accessory sperms per embryo and its development using this schedule. In total 116 German Holstein heifers received GnRH (i.m.; 0.05 mg Gonavet®, Veyx, Schwarzenborn, Germany) 60 h after PGF2α application (i.m.; 0.5 mg Cloprostenol forte®, Jenapharm, Jena, Germany) administered between Days 8 and 14 of the estrous cycle. Artificial insemination was carried out 13 h after GnRH application. Three different dosages of spermatozoa (15 × 106, 5 × 106, and 1 × 106) from three ejaculates from four fertile bulls were used. Embryos and oocytes were flushed from the oviducts of animals ovulated (n = 106; ovulation rate 91.3%). Animals were slaughtered on Day 4 after insemination. The quality of the embryos and oocytes was evaluated by microscopic examination. Embryos were stained with Hoechst 33258 to verify the number of accessory sperm. The evaluation of the data was carried out with the GLM procedure of the statistics software package SAS® (SAS Institute, Inc., Cary, NC, USA). As a post-hoc test the Student’s t-test was used. Significance was set at P = 0.05. After flushing of 106 animals, 85 embryos and oocytes was recovered (recovery rate 80.2%). Relative to the three sperm concentrations, there were no significant differences among fertilization rates (92.3, 96.2, and 78.8%) and among the portions of normal developed embryos (84.6, 80.7, and 75.8%, all respectively) between groups. Interestingly, significant differences were found according to the mean number of accessory sperm/embryo (29.6 ± 8.4, 45.3 ± 8.6, and 6.5 ± 7.2, respectively) and in the portion of embryos without or with more than 10 accessory sperm/embryo. Results show that fixed-time insemination, independent of detection of onset of estrus can result in high fertilization rates. Insemination with dosages <5 × 106 spermatozoa can reduce fertilization rates. Likewise, significant differences regarding fertilization rate were found after insemination of reduced sperm dosages of individual bulls. The number of accessory sperms/embryo seems to be an irrelevant parameter for quality of embryos produced under described conditions.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
B Rösing ◽  
A Bielfeld ◽  
J Neulen ◽  
P Habib

Abstract Study question Are inflammatory signatures in the blood of clinically asymptomatic patients predictive for oocyte fertilization success in ART treatment? Summary answer The proinflammatory M1 phenotype of macrophages combined with increasing numbers of cytotoxic T-cells and an upregulated inflammasome NLRC4 is associated with impaired oocyte fertilization. What is known already Oocyte fertilization is an indispensable step towards embryogenesis and reproductive success. Patients with acute or chronic inflammation are at risk of reduced fertilization rates and failure of assisted reproductive technology (ART) treatment. Inflammasomes are intracellular multiprotein complexes that mediate inflammatory tissue remodeling. They can be activated by endogenous and exogenous stimuli and result in maturation of the cytokines IL–1βand IL–18. Appropriate inflammasome activation serves for pathogen defense and tissue damage repair, while aberrant activation of inflammasomes can enhance uncontrolled tissue damage. Study design, size, duration This a prospective study including 39 patients stratified by fertilization rate (cut off 66%) of mature M II oocytes after ICSI procedure, compares 20 patients with high (mean 84,8%, range 67 –100%) versus 19 patients with low (mean 29.4%, range 0–65%) rates. Participants/materials, setting, methods We performed FACS analysis of immune cell composition (leukocytes, neutrophils, monocytes, macrophage types M1 and M2, cytotoxic T-cells, T-helper cells) and utilized RT-qPCR analysis of three inflammasomes (AIM 2, NLRP3 and NLRC4) and their down-streaming proteins caspase 1 and IL–1ß. We focused on an initial and a late state during controlled ovarian stimulation (COS) procedure. Main results and the role of chance This study reports a cellular and molecular inflammatory signature associated with reduced oocyte fertilization rates after ICSI treatment in clinically asymptomatic patients. On a cellular level proinflammatory M1 macrophages were significantly elevated in the low fertilization group (p &lt; 0.01), additionally the ratio of proinflammatory M1 macrophages to anti-inflammatory M2 phenotype was inversely associated with oocyte fertilization rate (p &lt; 0.001). Cytotoxic T cells (p &lt; 0.05) and the inflammasome NLRC4 (p &lt; 0.01) revealed identical patterns of association with fertilization rates in the group comparison. In line with this pattern, there was a significant upregulation of Caspase 1 with a consecutive increase in IL–1ß activity in women with low fertilization rates (p &lt; 0.05). No significant association was shown for the other tested immune cells (leukocytes, neutrophils, monocytes, T-helper cells) and inflammasomes (NLRC3, AIM2). Limitations, reasons for caution The preliminary results in this small study indicate an activation of the inflammasome NLRC4. This, however, has to be assessed on the protein level in a larger group of patients. Wider implications of the findings: Systemic macrophage augmentation of M1 phenotype and cytotoxic T cell presence in combination with upregulated inflammasome NLRC4 are associated with impaired oocyte fertilization. The predictive information of the identified immunologic signature could indicate targeted treatment to down regulate systemic inflammation and the use of oocyte activators in the ART laboratory. Trial registration number none


2005 ◽  
Vol 17 (2) ◽  
pp. 159 ◽  
Author(s):  
E.A. Martinez ◽  
J.M. Vazquez ◽  
I. Parrilla ◽  
C. Cuello ◽  
M.A. Gil ◽  
...  

A new procedure for nonsurgical deep intrauterine insemination (DUI) in non-sedated sows has recently been reported (Martinez et al. 2002 Reproduction 123, 163–170). In comparison to traditional artificial insemination (AI), using this procedure, a 20-fold reduction in the number of spermatozoa inseminated can be used without a decrease in fertility when hormonally treated post-weaning estrous sows are used. The aim of the present study was to evaluate the effectiveness of DUI under field conditions. In Experiment 1, crossbred sows (2–6 parity) were weaned at 20.75 ± 0.06 days. Estrous detection was performed once per day, beginning 3 days after weaning. Sows with a weaning to estrus interval of 4–5 days were selected to be inseminated. A total of 190 sows were inseminated at 12, 24, and 36 h after onset of estrus using one of the following two regimes: (1) DUI with 150 × 106 fresh spermatozoa in 5 mL of BTS (n = 95) and (2) Traditional AI with 3 × 109 fresh spermatozoa in 100 mL of BTS (n = 95) prepared from the same semen samples used for the DUI group. Farrowing rates (FR) and litter sizes (LTS; mean ± SEM) from both groups were compared using chi-squared test and ANOVA, respectively. There was no significant difference in the FR between groups (83.2 and 86.3% for DUI and AI groups, respectively). However, a decrease (P < 0.001) in the LTS was observed in sows inseminated by the DUI procedure (9.8 ± 0.29 and 10.9 ± 0.17, respectively). In Experiment 2, seventy one natural post-weaning estrus sows were used. Fifty-five sows were DUI inseminated three times with 150 (n = 17), 300 (n = 19), or 600 (n = 19) × 106 spermatozoa in 5, 10, or 20 mL of BTS, respectively. The remaining sows (n = 16) were traditionally inseminated. On Day 6 after estrus, sows were subjected to laparotomy and the tips of both uterine horns were flushed in order to evaluate pregnancy rate (PR: percentage of sows with at least 4 viable embryos) and fertilization rate (ratio of viable embryos to the total number of embryos and oocytes). PR was similar in all the groups, ranging from 84.2% (DUI 300 × 106 spermatozoa group) to 94.7% (DUI 600 × 106 spermatozoa group). Fertilization rate and the percentage of bilateral fertilization after DUI with 600 × 106 spermatozoa did not differ from those of the AI group (97.8 and 100% vs. 98.4 and 100%, respectively), but a significant decrease in both parameters (P < 0.05; chi-square test) was observed in sows inseminated with 300 (94.3 and 87.5%) or 150 (84.4 and 66.7%) × 106 spermatozoa. In conclusion, DUI with 150 × 106 spermatozoa offers similar FR but a lower LTS in sows with natural estrus in comparison with those parameters obtained when traditional AI is used. The lower litter size could be related to the low percentage of bilateral fertilization observed in that group. This work was supported by CDTI 020003.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Lópe. Ruiz ◽  
C Olmed. Illueca ◽  
M Bare. Gómez ◽  
S Roy. Bolea ◽  
L Aba. d. Velasco ◽  
...  

Abstract Study question Does Calcymicin improve reproductive outcomes of ICSI cycles in cases of fertilization failure and/or embryo blockage indications? Summary answer The application of the Calcymicin after ICSI improves reproductive outcomes, especially in cases with clinical indication of fertilization failure. What is known already According to the bibliography, deficiencies in the oocyte activation process frequently lead to failed ICSI cycles, and these can be corrected by increasing initial levels of calcium (Ca2+) in the oocyte using assisted oocyte activation techniques (AOA), such as the use of Ca2+ ionophores. Ca2+ Ionophores have been shown to trigger an initial Ca2+ spike in the ooplasm that activates Ca2+/Calmodulin dependent protein kinase II, which initiates the cascade of cellular events leading to oocyte activation. Previous results suggest that Ca2+ ionophore treatment can give live offspring after failed ICSI cycles. Study design, size, duration 270 oocytes collected from 17 patients who presented cycles with low fertilization rates and/or embryo blockage or poor quality embryos (according to ASEBIR’s embryo classification criteria) were retrospectively analyzed. Oocytes were divided into two groups, a control group that underwent conventional IVF/ICSI and another group that underwent an ICSI cycle with AOA. Study groups were defined according to clinical indications and subgroups according to AOA or control. All data were collected from 2017 until 2020. Participants/materials, setting, methods Among the 270 oocytes of the study sample, 142 belonged to the control group and 128 belonged to the AOA group. The AOA group oocytes were activated for 15 minutes immediately after ICSI using a prepared solution containing the Ca2+ ionophore A23187, CultActive© (Gynemed, Germany). Fertilization rate and type, blastocyst formation rate, blastocyst quality, embryo kinetics, and pregnancy rates were analyzed, all of them were compared to FIV/ICSI cycles without oocyte activation (control group). Main results and the role of chance In the analyses of the whole sample of oocytes, the AOA treatment gave a fertilization rate of 72.5%, which was significantly higher compared to 53.8% of the control cycles (p = 0.002). Good quality blastocysts and pregnancy rates were also significantly higher than the control (p = 0.01). In the group with an indication of fertilization failure, a significantly higher fertilization rate was recorded compared to the control (65% and 33%, respectively). A higher rate of abnormal embryos with three pronuclei was also found compared to the control (p &lt; 0.001). There were no significant differences in blastocyst formation rates, quality, or embryo kinetics (p &gt; 0.05). In the group with an indication of embryo blockage/poor embryo quality, a significantly higher rate of good quality blastocysts and lower blastulation time were recorded compared to the control (p &lt; 0.05). Limitations, reasons for caution The safety of the AOA technique with Ca2+ ionophore has not been fully demonstrated. In our study, none of the newborns had malformations, and gestational weeks and birth weights were normal. However, further studies on the safety of this technique are needed to implement it routinely in human reproduction clinics. Wider implications of the findings: According to these findings, an increase in the initial levels of calcium in the oocyte through the application of the Ca2+ ionophore A23187 after ICSI improves the results of failed assisted reproduction cycles, especially in the case of those diagnosed with fertility failure, which is a clear indication for AOA. Trial registration number Not applicable


2016 ◽  
Vol 14 (1) ◽  
Author(s):  
Eduardo Antônio Sanches ◽  
Danilo Caneppele ◽  
Renan Yoshiharu Okawara ◽  
Danielle Zanerato Damasceno ◽  
Robie Allan Bombardelli ◽  
...  

Abstract The Steindachneridion parahybae is an endangered catfish from Brazil and strategies applied for gametes optimization are necessary. The aim of this study was to assess inseminating doses and water volume upon the fertilization, hatching rates and percentage of normal larvae in S. parahybae . Was used a randomized design in factorial scheme (4×4) with four inseminating doses: 1.0×104, 1.0×105, 1.0×106, 1.0×107spermatozoa oocyte-1 and four volumes of water: 1, 35, 65 and 95mL of water g-1 of oocytes. The combination of doses and volumes were performed in triplicates (n=48). Each incubator (1.5L of useful volume) with 1g of oocytes was considered as an experimental unit. Significant interaction between inseminating doses and volumes of water to the values of the fertilization rates and quadratic effect of doses and volume for the values of hatching rates were observed. The doses and volumes did not influence the percentage of normal larvae (87.70±5.06%). It is recommended the use of 5.5×106 spermatozoa oocyte-1 and 1mL of water g-1 of oocytes during in vitro fertilization procedure. These results allowed us to develop new biotechnological strategies applied to the conservation of S. parahybae .


Calphad ◽  
2020 ◽  
Vol 69 ◽  
pp. 101762 ◽  
Author(s):  
A. Obaied ◽  
B. Bocklund ◽  
S. Zomorodpoosh ◽  
L. Zhang ◽  
R. Otis ◽  
...  

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