362 SEX RATIO OF IN VITRO-PRODUCED BLASTOCYSTS OBTAINED USING FROZEN SEMEN FROM A CLONED BULL OR ITS ORIGINAL CELL DONOR

2007 ◽  
Vol 19 (1) ◽  
pp. 296
Author(s):  
Y. Heyman ◽  
B. LeGuienne ◽  
C. Audouard ◽  
F. Charreaux ◽  
P. Humblot ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Vero Cells ◽  
Chi Square ◽  
Cell Donor ◽  
Frozen Semen ◽  
Chi Square Test ◽  
Original Cell ◽  
Vitro Development ◽  
Original Donor

Bulls obtained by somatic cell nuclear transfer (SCNT) have proved to develop normally, produce sperm, and be fertile (Shiga et al. 2005 Theriogenology 64, 334–343; Tecirlioglu et al. 2006 Theriogenology 65, 1783–1799). However, due to epigenetic variability encountered with clones, it cannot be excluded that major alterations may affect the X chromosome and especially the dosage of X inactivation in female embryos. This could result in a deviation of the sex ratio in offspring. To test this hypothesis, the sperm from a cloned bull was used in IVF to assess its ability to induce a different proportion of male and female embryos when compared to results obtained with the sperm of the original donor before cloning. In the present experiment, semen was collected twice weekly during 3 months from a 3-year-old cloned bull of the Charolais breed (H-2) and frozen-stored. Samples from 3 different ejaculates were used in comparison with control frozen straws from the original bull (H-0), prepared at the same age, to produce in vitro embryos. A total of 6 replicate IVF experiments were performed on the same batches of in vitro-matured oocytes (n = 654) using the standard swim-up and IVF technique in the laboratory. Twenty hours after insemination, presumptive zygotes were vortexed and cultured for 7 days in microdrops of B2 medium with Vero cells. Fertilization, cleavage, and blastocyst formation was assessed, and by Day 7, all of the blastocysts were individually frozen after grading. Two groups of 40 representative blastocysts derived from each bull (clone or donor) were used to determine the sex ratio using the sexing kit developed by UNCEIA R&D (Maisons-Alfort, France). Percentages between groups were compared by chi-square test. Semen from the cloned bull resulted in significantly lower fertilization and cleavage rates than that from the original donor (82.7% and 70% vs. 94.6% and 91%, respectively; P < 0.01), but further in vitro development was not different, as the proportions of blastocysts/cleaved were, respectively, 31.5% (74/235) vs. 38.7% (112/290). Sex determination was achieved in 79/80 of the in vitro-produced embryos and indicated that 55% of the blastocysts derived from the clone were male (22/40) and 45% female (18/40). This was not different from the proportion of male (61.5%, 24/39) and female (38.5%, 15/39) embryos in the group derived from semen of the original donor bull. In conclusion, these preliminary results indicate that the semen from this cloned Charolais bull has the same potential for in vitro embryo production as its original cell donor, and there is no evidence that SCNT could induce any deviation of the sex ratio. This observation needs to be confirmed with other sets of cloned bulls. (Shiga et al. 2005 Theriogenology 64, 334–343. Tecirlioglu et al. 2006 Theriogenology 65, 1783–1799.)

288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
M. Alomar ◽  
H. Tasiaux ◽  
S. Remacle ◽  
F. George ◽  
D. Paul ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test ◽  
Kinetics Of

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P < 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 � 9.5% vs. 21.9 � 6.7%; ANOVA: P < 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 � 2.1 hpi vs. 25.4 � 2.7 hpi for bull B; ANOVA: P < 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P < 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P < 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.

155 Fertility of sexed semen in Holstein heifers and cows

2020 ◽  
Vol 32 (2) ◽  
pp. 204
Author(s):  
M. Yamaguchi ◽  
M. Takayama ◽  
T. Nishisouzu ◽  
H. López ◽  
O. Dochi
Keyword(s):  
Sex Ratio ◽  
Dairy Cattle ◽  
Female Offspring ◽  
The Novel ◽  
Chi Square ◽  
Frozen Semen ◽  
Conception Rates ◽  
Sexed Semen ◽  
Parity Number ◽  
Chi Square Test

Compared with conventional semen, use of sexed semen decreases the conception rate (CR); therefore, it is important to address the factors that lead to sperm damage. Recently, a novel sexed semen technology was developed for improving dairy cattle fertility (Betthauser et al. 2016 J. Dairy Sci. 99 (E-Suppl. 1), 534). However, there are few reports about the fertility of this novel sexed semen in dairy cattle in Japan. The objective of this study was to compare the CR of traditional sorting semen (S1: flow cytometry/cell sorting) and the novel sexed semen (S2: SexcelTM, ABS Global Inc.) in Holstein heifers and cows in Japan. The CR for the first insemination was obtained from 391 Holstein cows and 148 heifers (10.7-17.8 months old) from 14 dairy farms in Hokkaido from June 2017 to April 2019. Semen used for AI was collected from six bulls and packaged into 0.25-mL straws and frozen. Cows inseminated with conventional frozen semen (S3: unsexed semen in 0.5-mL straws) were used as controls. Calving data were collected from 123 cows and 69 heifers that had calved between January 2018 and May 2019. The diagnosis of pregnancy was confirmed using ultrasonography between 30 and 45 days after insemination. The CR and sex ratio were analysed using chi-square test. The average parity number was 1.9±1.1, and average days open was 84.9±20.4 days. The average interval between calving and the first service was 86.2±20.3 days. The average milk yield (at 305 days) was 12 195±1595 kg. All of the animals were inseminated after the onset of standing heat or removal of the tail chalk of natural heats and oestrus and ovulation synchronisation programmes on the farm. Conception rates of heifers of the first service of S1 (n=54) and S2 (n=94) were 68.5 and 70.2%, respectively. Conception rates of cows of the first service of S1 (n=32), S2 (n=173), and S3 (n=186) were 56.3, 57.8, and 53.8%, respectively. There were no significant differences in CR between these groups (P>0.4). The proportion of female calves produced using S1 (n=61) and S2 (n=57) was 91.8 and 93.0%, respectively, which was significantly more (P<0.01) compared with the 50.0% produced using S3 (n=74). There were no significant differences between S1 and S2 (P>0.8) in terms of the proportion of calves produced. The results of this study demonstrated that the traditional sorting and novel sexed semen (S1 and S2, respectively) did not differ in terms of the first-service CR and produced considerably more female calves than when the conventional semen (S3) was used. Furthermore, these findings indicate that the use of this novel sexed semen could achieve a sex ratio close to 90% female offspring.

214 EFFECT OF LONG-DISTANCE TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED - IN VITRO-CULTURED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 206
Author(s):  
M. Kanae ◽  
K. Hisaichi ◽  
S. Jaswant ◽  
D. Osamu
Keyword(s):  
Calf Serum ◽  
Control Group ◽  
Long Distance ◽  
Chi Square ◽  
Maturation Rate ◽  
Long Time ◽  
Chi Square Test ◽  
Thermos Flask ◽  
Vitro Development

Keeping ovaries for a long time before oocyte recovery affects the maturation rate and blastocyst rate (BL rate), but a change in the temperature of the ovary at the time of transportation does not affect the BL rate (Ribeiro et al. 2008 Anim. Reprod. Sci. 108, 171–179). The objective of this study was to investigate the effect of long-distance transportation of ovaries on the in vitro development of bovine oocytes into blastocysts. Ovaries in the transportation group (Group T) were collected from a slaughterhouse, placed inside a Thermos flask, and transported to the laboratory within 17 to 21 h. The Thermos flask was covered with a freezer pack in a foam polystyrene box (Nakatate et al. 2006 Reprod. Fertil. Dev. 18, 193). The temperature of the Thermos flask was measured with a temperature recorder. Cumulus–oocyte complexes (COC) were collected by aspirating 2- to 6-mm follicles from the ovaries. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates, CL rate) and on Days 7 to 9 (BL rate). The ovaries in the control group (Group C) were collected from a slaughterhouse different from that of the Group T ovaries and transported to the laboratory in saline (23°C) in a Thermos flask within 3 h. These ovaries were then used for the collection of COC in the same way as for Group T. Data were analysed by chi-square test. A total of 25 experiments were performed using the ovaries from Groups C and T (n = 3945 v. n = 7218). The CL rate and the BL rate for Group T were significantly lower than those for Group C (41.8 v. 62.0% and 16.1 v. 29.7%; P < 0.01). In Group T, the duration of ovary transportation did not affect the CL rate (within 19 h, between 19 and 20 h, and more than 20 h), but the BL rate was significantly decreased with an increase in transport time (18.7 v. 16.1 v. 14.8%). The temperature change in the Thermos flask during transportation ranged from 12.0 to 25.1°C; the temperature was maintained virtually constant or decreased. The CL rates of the groups that underwent a temperature (t) change (t ≤ 5.0 and 5.0 < t ≤ 10.0) were not significantly different, but the BL rate in the group with 5.0 < t ≤ 10.0 was significantly lower than that of the other group (16.7 v. 13.5%; P < 0.01). These results suggest that the long-distance transportation of ovaries affects in vitro embryo development. Moreover, a decrease in temperature during transportation may have an adverse effect on blastocyst formation.

298 THE INFLUENCE OF DISTINCT CENTRAL BULL STATIONS ON THE IN VITRO EMBRYONIC DEVELOPMENT OF NELORE BULLS

2010 ◽  
Vol 22 (1) ◽  
pp. 305
Author(s):  
A. Renzi ◽  
F. P. Elias ◽  
R. A. Vila ◽  
R. B. Lôbo
Keyword(s):  
Calf Serum ◽  
Cumulus Cells ◽  
In Vitro Production ◽  
Freezing And Thawing ◽  
Chi Square ◽  
Frozen Semen ◽  
Chi Square Test ◽  
Vitro Production ◽  
In Vitro Embryonic Development

Reproductive biotechnology is growing worldwide as one of the most important tools of cattle breeding because it accelerates the process of genetic improvement. Most of the embryos produced are obtained using frozen semen from different AI centers. During freezing and thawing of semen, the sperm can be damaged by the rapid and dramatic changes in the physicochemical conditions that occur during cooling and ice formation. It has previously been described that bad management of frozen semen can result in reduced fertilization. This study investigated the influence of different central bull stations on the development of in vitro-produced bovine embryos. We compared semen of 154 Nelore bulls, used for IVF, from 8 different central bull stations (all of which used the same cryopreservation protocol) in the development of blastocysts. The in vitro production of embryos was performed as described: oocytes were collected from the slaughterhouse and matured in TCM-199 + 10% fetal calf serum (FCS) +0.5 μg mL-1 FSH + 50 μg mL-1 LH+ 1 μg mL-1 estradiol, for 24 hat 38.5°C in 5%CO2 in atmospheric air. Viable spermatozoids were obtained by centrifugation in Percoll gradient (45 and 90%), and used for IVF in a concentration of 2 million spermatozoa per mL in TALP + 10 μg mL-1 of heparin medium. After 12 h, the presumptive zygotes were transferred to a CR2+ 10% FCS medium and co-cultured with cumulus cells. After 168 h of IVF, we evaluated the number and stage of cleaved embryos produced with the semen of each bull. Statistical analyses were performed by using the chi-square test. Our results suggest that there are differences among distinct central bull stations in the proportion of embryos that developed into blastocysts and the different stages they hatched. FAPESP, CNPq, PROEX, FAEPA.

349 EFFECT OF CUMULUS CELLS AND MATURATION CULTURE PERIOD OF OOCYTES ON THE SEX RATIO OF IN VITRO-FERTILIZED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 331 ◽  
Author(s):  
G. Z. Mingoti ◽  
B. C. S. Leão ◽  
F. P. Gottardi ◽  
R.-V. Alonso
Keyword(s):  
Sex Ratio ◽  
Cumulus Cells ◽  
Culture Period ◽  
Blastocyst Development ◽  
Chi Square ◽  
Maturation Medium ◽  
Chi Square Test ◽  
Culture Duration ◽  
The Difference

Studies have reported a high proportion of males from IVP of embryos systems. Factors such as the stage of oocyte maturation at the time of insemination and conditions of the IVC may influence the proportion of sex. Although cumulus cells (CC) improve the fertilization ability of sperm cells, there are some evidence that exposure of sperm to CC increases the proportion of male embryos produced in vitro. The objective of this study was to investigate the effects and interaction between CC and IVM culture period of bovine oocytes on the sex ratio of in vitro-produced embryos. COCs (n = 781) were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (18, 20, 22, and 24 h). Maturation medium consisted of TCM-199 with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL-1 gentamicin, 0.5 μg mL-1 FSH, 100 IU mL-1 hCG, and 10% FBS. After maturation culture, oocytes were inseminated with frozen-thawed spermatozoa. Oocytes were inseminated included in CC (COC) or denuded of such cells (denuded oocytes, DO). All zygotes were then co-cultured in vitro with CC in SOF medium with 0.5% BSA and 2.5% FBS. Cultures were carried out at 38.5°C and 5% CO2 in air. Cleavage and blastocyst development rates were observed, respectively, at 48 (Day 2) and 168 (Day 7) h post-insemination (hpi). Blastocysts were harvested on Day 7, and the sex of the embryos was examined using the pair of primers S4B (Kageyama S et al. 2004 Theriogenology 66, 509-514). The data (mean ± SEM) were analyzed by ANOVA, in which the effects of the existence of CC (COC v. DO) and IVM culture periods (18 h, 20 h, 22 h v. 24 h), and interaction between these variables were evaluated. Multiple comparisons of means were followed using Tukey’s test (P < 0.05). The chi-square test was utilized to test the difference in the proportion of sex of embryos. Independently of the presence or absence of CC during IVF, there was no effect of IVM culture duration (P > 0.05) on cleavage (81.5 to 94.8% for COC; and 71.7 to 75.8% for DO) and blastocyst rates (20.0 to 35.9% for COC; and 10.0 to 15.5% for DO). But independently of IVM culture duration, the comparison between COC and DO groups revealed that absence of CC adversely affected (P < 0.05) the cleavage (87.0 and 73.3%, respectively; averaged results) and blastocyst rates (24.5 and 13.5%, respectively; averaged results). In COC, the proportion of male blastocysts increased with the progression in maturation culture period from 18 up to 22 h (54.2, 65.9, and 83.3%, respectively). But for DO, the proportion of females was higher, with exception of 22 h IVM group (65.4, 32.3, 53.3, and 60.0%, respectively from 18 to 24 h of IVM). These results indicate that the sex ratio of in vitro-fertilized embryos is apparently influenced by the maturation culture period of the oocytes and by the presence of CC. FAPESP.

132 TIMING OF THE FIRST ZYGOTIC CLEAVAGE WAS NOT RELATED TO THE SHIFT IN SEX RATIO OF BOVINE BLASTOCYST IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 165
Author(s):  
K. Zaorska ◽  
E. Pers-Kamczyc ◽  
D. Lechniak
Keyword(s):  
Sex Ratio ◽  
Positive Influence ◽  
Control Group ◽  
Fish Analysis ◽  
Chi Square ◽  
Experimental Conditions ◽  
Female Ratio ◽  
Developmental Potential ◽  
Frozen Semen

It has been shown that embryos which cleaved earlier (<30 h postinsemination, hpi) have greater chance to develop to blastocyst. It has been shown that in in vitro conditions male embryos develop faster than females. Further, spermatozoa with the Y chromosome more frequently penetrate oocytes during the first 6 hpi. The aim of this experiment was to investigate the sex-related embryo growth rate in relation to the timing of the first zygotic cleavage and GH presence during IVM. Oocytes were matured in TCM-199 supplemented with fatty acid free BSA and hormones (FSH and GH) and then inseminated (Parrish et al. 1998 Biol. Reprod. 38, 1171–1180), whereas embryos were cultured in sequential media (Lane et al. 2005 Theriogenology 60, 407–419). All embryos that cleaved by 30 hpi (early cleavers, EC) were selected and cultured separately. The remaining embryos cleaved by 48 hpi (non early cleavers, NEC) were also incubated in separate drops. Blastocysts of proper morphology were collected at 176 hpi and subjected to sex determination by PCR (AMGL gene). The significance of developmental stage, timing of the first zygotic cleavage and GH supplementation in relation to the sex ratio was evaluated by the chi-square test. All straws with frozen semen of 2 bulls used in this experiment were derived from single ejaculates. The sex ratio of sperm samples used for IVF was evaluated by FISH with locus-specific probes. The experiment was done on 266 embryos obtained from 1249 oocytes. A significant predominance of male blastocysts regardless of the experimental conditions was observed [the male to female ratio (M:F) 2:1, P < 0.001]. FISH analysis revealed that there was no deviation from the expected 1:1 ratio of X and Y spermatozoa in sperm samples used for IVF. When the timing of the first zygotic cleavage was considered, shift in M:F ratio in favor of males (P < 0.01) in blastocysts derived from EC zygotes was noticed. Due to a very low number of NEC embryos, this category was not included in analysis. Although the M:F ratio was shifted towards males, the rate of female embryos was greater in the control group (25F/38M) v. GH group (16F/39M) of EC expanded blastocysts. This phenomenon was not observed among hatched blastocysts; however, GH presence caused an increase (P < 0.01) in the number of females at this stage. Therefore GH may stimulate embryonic growth, especially embryos of reduced quality, through its positive influence on cytoplasmic oocyte maturation. In conclusion, the predominance of male blastocysts observed in this study may be attributed to the applied IVF procedure because the X:Y ratio in spermatozoa was not different from the expected 1:1 ratio. Moreover, significantly fewer females among analyzed blastocysts may suggest that the developmental potential of female embryos in applied in vitro conditions was somehow reduced when compared with males. This became evident when the transition from expanded to hatched blastocysts was observed. This work was supported by the project No. N302 046 31/3780 of the Ministry of Science and Higher Education, Poland.

194 Superovulation response does not affect embryo development of pronuclear microinjected embryos in the goat

2019 ◽  
Vol 31 (1) ◽  
pp. 221
Author(s):  
N. Buzzell ◽  
S. Blash ◽  
K. Miner ◽  
M. Schofield ◽  
J. Pollock ◽  
...  
Keyword(s):  
Fertilization Rate ◽  
Developmental Competence ◽  
Variable Response ◽  
Chi Square ◽  
P Values ◽  
Chi Square Test ◽  
Multiple Donors ◽  
Vitro Development ◽  
Short Timeframe

Superovulation of donor animals is essential in the production of transgenic founder goats generated through microinjection. There can be a marked variable response to the exogenous hormones used for superovulation. The objective of this study was to examine how the superovulatory response of individual goats affected the ability of the fertilized, microinjected embryos to develop into offspring. The donors were superovulated using a progesterone implant on Day 0, a prostaglandin injection at Day 7, 2 injections ~12h apart of 32 to 36mg of FSH on Day 12 to 15, progesterone implant removal on Day 14, bred by intact bucks several times starting on Day 15 to 16, an injection of 50μg of gonadotropin-releasing hormone, and surgical collection of 1- to 2-cell embryos from retrograde flushing of the oviduct on Day 17 (~24-48 h, 1-2 days after breeding). Surgical collection allows for an accurate ovulation point (OP) count before the oviduct being retrograde flushed and ova collected and counted. Data from donor animals were grouped by superovulatory response based on OP counts of 1-10, 11-20, 21-30, or &gt;30. The number of donors that contributed per group were 130, 280, 175, and 52, respectively. The recovery rate was 76, 72, 68, and 62%, respectively. After collection, ova were viewed under a dissecting microscope and assessed for fertilization by identifying pronuclei, and 1 pronucleus was microinjected. The fertilization rate was 47, 52, 51, and 56%, respectively. The survivability rate after microinjection was 80, 76, 75, and 76%, respectively. Surviving embryos were transferred (3-5) into recipient goats following a 2- to 6-h in vitro culture (as 1- to 2-cell embryos), allowing for a suitable period to assess viability post-injection. Further in vitro development rates were not assessed because of the short timeframe the embryos stayed in culture. The conception rates were 71, 56, 65, and 53%, respectively, and abortion rates were 23, 10, 14, and 9%, respectively. As some recipients received embryos from multiple donors, this data could not be included in the analysis as identifying which offspring were from the corresponding embryo group could not be confirmed. Data were analysed using SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). The Wald chi square test under linear regression was used to analyse the number of offspring produced per embryo transferred. No significant differences were found between groups (all P-values were&gt;0.05). This analysis indicated that the range of superovulation response does not affect the developmental competence of the pronuclear microinjected embryo or the ability to produce viable offspring. Table 1.Comparison of the donor ovulation counts, number of embryos transferred, offspring produced and overall efficiency

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals Comparison of bacterial microleakage of three bioactive endodontic sealers in simulated underwater diving and aviation conditions

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mehdi Dastorani ◽  
Behnam Malekpour ◽  
Mohsen AminSobhani ◽  
Mohammadsadegh Alemrajabi ◽  
Arezoo Mahdian ◽  
...  
Keyword(s):  
Atmospheric Pressure ◽  
Mineral Trioxide Aggregate ◽  
In Vitro Study ◽  
Chi Square ◽  
Custom Made ◽  
Single Cone ◽  
Registration Number ◽  
Chi Square Test ◽  
Pressure Changes

Abstract Background Bacterial microleakage is an important cause of apical periodontitis and endodontic treatment failure. This study aimed to assess the bacterial microleakage of nano-mineral trioxide aggregate (nano-MTA) as a sealer, Endoseal MTA, and GuttaFlow Bioseal sealers in atmospheric pressure, and simulated underwater diving and aviation conditions. Methods In this in vitro, experimental study, 180 extracted single-rooted teeth were cleaned and shaped, and were then randomly divided into three groups for single-cone obturation using Endoseal MTA, GuttaFlow Bioseal, or nano-MTA as a sealer. Each group was then randomly divided into three subgroups, and subjected to ambient atmospheric pressure, 2 atm pressure (to simulate underwater diving), and 0.5 atm pressure (to simulate aviation) using a custom-made pressure chamber. The teeth then underwent microbial leakage test using Streptococcus mutans (S. mutans), and the percentage of samples showing microleakage was recorded for up to 1 month, and analyzed using the Chi-square test. Results The three sealer groups were significantly different regarding bacterial microleakage (P < 0.05). The nano-MTA group showed significantly higher microleakage after 15 days than the other two groups (P = 0.006). The effect of pressure on bacterial microleakage was not significant in any sealer group (P > 0.05). Conclusion Within the limitations of this in vitro study, it may be concluded that single-cone obturation technique using nano-MTA as a sealer results in lower resistance to bacterial microleakage compared with the use of GuttaFlow Bioseal, and Endoseal MTA. Pressure changes in simulated underwater diving and aviation conditions had no significant effect on bacterial microleakage. Trial Registration Number This is not a human subject research.

scholarly journals 252 COMPARISON OF TWO SPERM SEPARATION PRODUCTS FOR USE IN BOVINE IVF

2005 ◽  
Vol 17 (2) ◽  
pp. 276 ◽  
Author(s):  
J. Pryor ◽  
S. Romo ◽  
D.D. Varner ◽  
K. Hinrichs ◽  
C.R. Looney
Keyword(s):  
Sperm Concentration ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Before And After ◽  
Chi Square Test ◽  
Group 2 ◽  
Live Sperm ◽  
Sperm Separation

In commercial bovine in vitro fertilization (IVF) companies, there is a continuous need to improve results. Efforts to maximize in vitro embryo production have included modifications in the use of sperm separation gradients. The development of commercially available sperm centrifugation gradients represents a new possibility of increasing the number of viable sperm that can be obtained from low concentration (fresh or frozen, sexed or unsexed) semen samples in order to improve the efficiency of the IVF system to make embryo production as efficient as possible. The objective of this study was to compare two different separation gradients, as follows: Group 1: Percoll (Sigma, St. Louis, MO, USA), in 45% and 90% gradients; Group 2: EquiPure (Nidacon, Gathenburg, Sweden), in top and bottom layers. Before and after separation, sperm were evaluated at 200× magnification for total motility, and then stained to assess viability at 400× with fast-green/eosin stain (Sigma). Sperm separation was performed using frozen/thawed semen from one bull. Semen was separated by centrifugation at 200g for 30 min in both density gradients. Results obtained from Groups 1 and 2 were compared by chi-square test. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 92 × 106, vs. 159 × 106 sperm/mL for EquiPure; P < 0.05) but resulted in higher motility (60% vs. 39%, respectively; P < 0.05) of separated sperm. Rates of live sperm cells were not significantly different between groups (69.5% vs. 70%, respectively; P > 0.1). These results indicate that the commercial separation medium EquiPure may be associated with higher sperm concentration levels but with lowered sperm motility when compared to Percoll for bovine sperm separation. However, Equipure provided similar percentages of live sperm when compared to Percoll, which is currently used in our laboratory.

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