Evaluation of chromatin integrity of motile bovine spermatozoa capacitated in vitro

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Z. Reckova ◽  
M. Machatkova ◽  
R. Rybar ◽  
J. Horakova ◽  
P. Hulinska ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Production Efficiency ◽  
Sperm Chromatin ◽  
Percoll Gradient ◽  
Embryo Production ◽  
Before And After ◽  
Bovine Spermatozoa ◽  
Chromatin Integrity ◽  
The Mean

SummaryThe efficiency of in vitro embryo production is highly variable amongst individual sires in cattle. To eliminate that this variability is not caused by sperm chromatin damage caused by separation or capacitacion, chromatin integrity was evaluated. Seventeen of AI bulls with good NRRs but variable embryo production efficiency were used. For each bull, motile spermatozoa were separated on a Percoll gradient, resuspended in IVF–TALP medium and capacitated with or incubated without heparin for 6 h. Samples before and after separation and after 3-h and 6-h capacitacion or incubation were evaluated by the Sperm Chromatin Structure Assay (SCSA) and the proportion of sperm with intact chromatin structure was calculated. Based on changes in the non-DFI-sperm proportion, the sires were categorized as DNA-unstable (DNA-us), DNA-stable (DNA-s) and DNA-most stable (DNA-ms) bulls (n = 3, n = 5 and n = 9, respectively). In DNA-us bulls, separation produced a significant increase of the mean non-DFI-sperm proportion (p ≤ 0.01), as compared with the value before separation. Capacitacion produced a significant decrease in the mean non-DFI-sperm proportion in H+ sperm (p ≤ 0.01). In DNA-s bulls, separation significantly increased the mean non-DFI-sperm proportion (p ≤ 0.01) but during capacitacion, the mean non-DFI-sperm proportion remained almost unchanged. In DNA-ms bulls, neither separation nor capacitacion had any effect on the mean non-DFI-sperm proportion. It can be concluded that, although separation and capacitacion may produce some changes in sperm chromatin integrity, these are not associated with different in vitro fertility of the bulls involved.

scholarly journals Monitoring of chromatin integrity changes in the population of motile bovine sperm capacitated in vitro

2007 ◽  
Vol 55 (2) ◽  
pp. 65-70
Author(s):  
Zuzana Rečková ◽  
Marie Machatková ◽  
Roman Rybář ◽  
Ladislav Máchal
Keyword(s):  
Chromatin Structure ◽  
Gradient Centrifugation ◽  
Percoll Gradient ◽  
Integrity Assessment ◽  
Bovine Sperm ◽  
Significant Difference ◽  
Before And After ◽  
Chromatin Integrity ◽  
The Mean

The objective of our study was to standardize a method for chromatin integrity assessment in a separated population of bovine sperm and monitor the changes occurring during sperm capacitation stimulated with heparin. Frozen sperm of 11 young bulls of the Czech pied breed with a defined fertility in both in vitro system (from 12.9% to 25.8% embryos) and in insemination (from 60.2% to 66.4% pregnancy) was used in our experiments.Bovine spermatozoa were isolated by Percoll gradient centrifugation from frozen-thawed semen using Tyrode’s medium (SP-Talp) and resuspended in a fertilization medium (IVF-Talp). The spermatozoa were incubated at laboratory temperature at a concentration 25 × 106 per cm3 for 6 h either in IVF-Talp medium with heparin (H+) or without heparin (H–). Samples were obtained immediately after sperm thawing (PS), following motile spermatozoa separation (P0), and their three (P3) and six hour (P6) incubation. The samples were examined by flow cytometry. Two measurements were carried out in each of the samples so that a total of 10 thousand spermatozoa were analysed. Proportion of spermatozoa with undetectable DNA fragmentation index (non-DFI sperm) i.e. spermatozoa with undamaged chromatin structure were determined using SCSA-soft software.Chromatin integrity changes of spermatozoa before and after separation and capacitation differed markedly in individual bulls. Separation of motile spermatozoa increased significantly the mean proportion of non-DFI sperm in tested bulls (from 94.2 to 96.4%, P ≤ 0.01). While in most of the bulls the mean proportion of non-DFI sperm remained nearly constant during incubation (H–) (mean, P0 – 96.4%, P3 – 95.6%, P6 – 95.5%), it gradually decreased during capacitation (H+) (mean, P0 – 96.4%, P3 – 95.2%, P6 – 94.2%). The differences were statistically significant (P0 vs. P3H+, P0 vs. P6H+, P ≤ 0.05). Significant difference (P ≤ 0.05) in the mean proportion on non-DFI sperm was also found between capacitated (P6H+) and incubated (P6H–) spermatozoa.The results of our study suggest the following outcomes. Separation of motile spermatozoa by Percoll gradient increased the proportion of spermatozoa with undamaged chromatin structure. Sperm incubation induced gentle damage of chromatin integrity which was potentiated by heparin in capacitated spermatozoa. The proportion of spermatozoa with undamaged chromatin structure remained relatively high in the course of capacitation, therefore we can assume it to be high enough for a potential oocyte fertilization.

scholarly journals Effect of quality of sperm chromatin structure on in -vitro production of cattle embryos

2005 ◽  
Vol 48 (1) ◽  
pp. 32-39
Author(s):  
L. Kątska-Książkiewicz ◽  
M. Bochenek ◽  
B. Ryńska
Keyword(s):  
Chromatin Structure ◽  
Blastocyst Stage ◽  
Sperm Chromatin ◽  
In Vitro Production ◽  
Embryo Production ◽  
In Vitro Embryo Production ◽  
Bull Ejaculate ◽  
Vitro Production

Abstract. Bull effect on results of in vitro embryo production has been well documented. The aim of the present study was to find the relationship between quality of bull sperm chromatin and its effect on in vitro embryo production. Bovine in vitro matured oocytes were fertilized in vitro using capacitated spermatozoa (freshly ejaculated or frozen-thawed) of 12 bulls. Semen was simultaneously processed according to the sperm chromatin structure assay (SCSA) method and was analysed by flow cytometry. At least 3 replications of IVP with the same semen sample were done. The percentage of spermatozoa with abnormal chromatin ranged from 0.4% to 23.8%. All bulls used for the experiment were divided into three groups showing minimal (0.82% ± 6.82%), low (1.70% ± 15.82%) and high (18.16% ± 53.59%) percentages of spermatozoa with abnormal chromatin structure. Both cleavage rates and embryo development to the blastocyst stage were correlated significantly with sperm chromatin abnormalities and resulted in 23.1, 17.7 and 12.2% of blastocysts respectively for sperm with minimal, low and high percentages of chromatin abnormalities. The SCSA method may be used as a practical indicator of suitability of bull ejaculate for IVP purposes.

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.

212 EFFECT OF CONSECUTIVE SUPERSTIMULATORY TREATMENT-INDUCED FOLLICULAR WAVE SYNCHRONIZATION TO OPTIMIZE OOCYTE RETRIEVAL AND EMBRYO PRODUCTION BY OVUM PICKUP AND IN VITRO FERTILIZATION IN COWS

2011 ◽  
Vol 23 (1) ◽  
pp. 205
Author(s):  
K. Imai ◽  
M. Ohtake ◽  
Y. Aikawa ◽  
S. Sugimura ◽  
M. Hirayama ◽  
...  
Keyword(s):  
Development Program ◽  
Oocyte Retrieval ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Follicular Wave ◽  
The Third ◽  
The Mean ◽  
Student’S T

We previously reported that superstimulatory (SS) treatment-induced follicular wave synchronization after ovum pickup (OPU) was effective in enhancing the quality of obtained oocytes and blastocysts derived from in vitro maturation (IVM) and fertilization (IVF; Imai et al. 2010 Reprod. Fertil. Dev. 22, 296). The present study was designed to examine the efficiency of embryo production by 4 sessions of OPU-IVF using a series of the SS treatment-induced follicular wave synchronizations. For the SS protocols, 3 consecutive SS (3CSS) and 2 separated SS (2SSS) were used. In the 3CSS group, the first OPU was performed on random days of the oestrous cycle (Day 0) and all follicles larger than 2 mm in diameter were aspirated. On Day 5, follicles larger than 8 mm in diameter were aspirated and a CIDR (InterAg, Hamilton, New Zealand) was inserted. The cows then received 20 armour units of FSH (Kawasaki-Seiyaku, Kawasaki, Japan) in twice-daily decreasing doses by IM injection from Day 7 to 10. Cloprostenol (PGF; 0.75 mg, Fujita-Pharm, Tokyo, Japan) was administered on the morning of Day 9. The second OPU was performed 48 h after PGF administration on Day 11; the CIDR was removed from the cows just before OPU. After the second OPU, donors were treated consecutively with the SS protocol mentioned above for the third and fourth OPU sessions. In the 2SSS group, donors received 2 sets of the SS treatment mentioned above, with an interval of 11 days between the second and the third OPU session. Four OPU sessions were performed every 11 days on all cows. In this study, 8 Japanese Black cows were divided into the 3CSS and 2SSS groups, and the treatment for each group was reversed after a 65-day interval as crossover trials. After OPU, Grade 1 and 2 oocytes were used for IVM and IVF, and putative zygotes were cultured as described by (Imai et al. 2006 J. Reprod. Dev. 52, S19–S29 suppl.). A part of the zygotes were cultured in a micro-well system. Data were analysed by Student’s t-test and chi-square test. There were differences (P < 0.05) in the mean (±SD) number of follicles, collected oocytes, and cultured oocytes in the 3CSS (35.0 ± 8.6 and 24.4 ± 11.2, respectively) and 2SSS (30.8 ± 10.5 and 20.2 ± 9.0, respectively) groups. There were no differences in mean percentage of blastocyst formation and Grade 1 blastocyst rates between the 3CSS (38.5 and 55.8%, respectively) and 2SSS (34.8 and 54.8%, respectively) groups. However, the mean number of blastocysts produced per OPU session was significantly (P < 0.05) higher in the 3CSS group (8.1 ± 6.3) compared with the 2SSS group (5.8 ± 4.4). These results indicate that a series of 3 consecutive SS treatments had greater efficiency in producing OPU-IVF embryos. This work was supported in part by the Research and Development Program for New Bio-industry Initiatives.

206 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON IN VITRO EMBRYO PRODUCTION

2008 ◽  
Vol 20 (1) ◽  
pp. 182 ◽  
Author(s):  
K. Imai ◽  
Y. Inaba ◽  
H. Yoshioka ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Annual Conference ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Follicular Wave ◽  
The Mean ◽  
In Vitro Embryo Production

We previously reported that follicular wave synchronization, by removal of the dominant follicle on Day 5 after ovum pickup (OPU), was effective in increasing oocyte quality in the developing follicles (Imai et al. 2006 32th Annual Conference of the IETS, poster presentation no. 277). The current study was designed to examine the effect of superstimulatory treatment to induce subsequent follicular wave synchronization on embryo production by OPU and IVM-IVF-IVC in Holstein dry cows. Cows were reared under the same feeding and environmental conditions, and 2 OPU sessions were conducted in each cow. In the first session, OPU was performed in 8 cows on arbitrary days of the estrous cycle by using a 7.5-MHz linear transducer with needle (Cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, Aloka, Tokyo, Japan). Follicles larger than 8 mm in diameter were then aspirated and a CIDR was inserted on Day 5 (the day of first OPU session = Day 0). Cows then received 30 mg of FSH (Antrin-R10; Kawasaki Mitaka Pharmaceutical Co., Tokyo, Japan) twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by i.m. injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). The second OPU session was performed 48 h after PGF administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. Grades 1 and 2 COC were matured, fertilized, and cultured as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. Embryo development was assessed by the cleavage rate on Day 2 and by the blastocyst formation rate on Days 7 to 8 (the day of insemination = Day 0). Data were analyzed by Student's t-test. There were no differences in the mean (� SD) number of aspirated follicles or collected oocytes between the first (32.5 � 6.8 and 26.0 � 12.7, respectively) and second (29.3 � 10.4 and 19.0 � 9.4, respectively) OPU sessions (P > 0.1). The percentage of Grade 1 and 2 oocytes for the second OPU session (90.5 � 13.8%) was significantly higher (P < 0.01) than for the first OPU session (63.1 � 6.3%), and significant differences were found for cleavage (79.4 � 14.1, 61.8 � 25.1, P < 0.01) and blastocyst rates (68.1 � 16.7, 24.2 � 22.3, P < 0.001) between sessions. The mean numbers of blastocysts obtained per session were 4.3 � 2.9 and 12.8 � 8.7 in the first and second sessions, respectively (P < 0.01). These results indicate that superstimulatory treatment and subsequent follicular wave synchronization were effective on in vitro embryo production by increasing the oocyte quality.

361 INFLUENCE OF SEXING PROCEDURE ON BULL SPERM CHROMATIN STRUCTURE

2007 ◽  
Vol 19 (1) ◽  
pp. 296
Author(s):  
M. Bochenek ◽  
T. Herjan ◽  
Z. Smorag
Keyword(s):  
Chromatin Structure ◽  
Laser Power ◽  
Sperm Chromatin ◽  
Control Group ◽  
Uv Laser ◽  
Sexed Semen ◽  
The Mean ◽  
Bull Sperm ◽  
Group 2 ◽  
Control Samples

Flow cytometry is the only reliable and relatively fast method allowing separation of live X and Y spermatozoa for sex regulation. Many thousands of animals of different mammalian species have been born after insemination with sexed semen during the past 20 years. Nevertheless, the question is still open: does the bull sperm sexing technology affect chromatin structure? A case of serious chromatin damage after sexing stallion semen was reported previously (Bochenek et al. 2006 Havemeyer Foundation Monograph Series No. 18, 13 –14). The aim of this work was to examine the effect of the sexing procedure and different UV laser powers on bull sperm chromatin structure. The ejaculates of 28 bulls (one ejaculate/bull) were used in the study. Each ejaculate was divided into 5 groups: (1) control, unprocessed; (2) sorted strictly according to XY Inc. protocol (Schenk et al. 1999 Theriogenology 52, 1375 –1391); (3) as group 2, but without the Red Food dye staining used for dead spermatozoa discrimination; (4) as group 2, but with double UV laser power (300 mW); and (5) as group 3, but with double UV laser power (300 mW). Sperm sorting was performed with a MoFLoSX flow cytometer at speeds of 3000 –5000 cells/s. Sorted fractions of X and Y spermatozoa were mixed again and stored for 24 h at 15 °C. A sperm chromatin structure assay (SCSA) was performed twice on each sorted sample, immediately after sorting and after 24 h. The chromatin of control samples was examined according to the same time schedule. The percentage of spermatozoa with damaged chromatin was calculated (COMP α-t) as well as standard deviation of the α-t parameter (SD α-t). The latter parameter, although less intuitive, is considered as even more precise than COMP α-t in chromatin investigations. The mean percentage of spermatozoa with abnormal chromatin was 1.12% (SD = 0.47) for control samples. The highest level of chromatin abnormality was noted for the 300 mW group with no dead cell discrimination (Red Food staining): 1.29% (SD = 1.05). After 24 h of storage, the mean level of chromatin abnormality increased to 1.97% (SD = 0.96) in control samples whereas that in all sorted samples was lower: from 1.06% (SD = 0.4) to 1.16% (SD = 0.62) in the 150 mW/non-Red Food-stained and the 300 mW/Red Food-stained groups, respectively. This difference appeared to be statistically significant (t; P ≤ 0.05). Interestingly, the percentage of abnormal spermatozoa decreased slightly after 24 h of storage in the 300 mW/Red Food-stained and the 300 mW/non-Red Food-stained groups ( –0.13% and –0.08%, respectively). Calculation of the SD α-t parameter showed statistically significant differences in chromatin abnormality between the control group vs. the 300 mW/non-Red Food-stained group immediately after sorting and the control group vs. the 150 mW/Red Food-stained group after 24 h of storage. In conclusion, although the statistically significant increase of chromatin damage was found after sexing in some investigated groups, it seems that the level of this abnormality is far too low to affect sexed semen fertility.

281 EVALUATION OF IN VITRO EMBRYO PRODUCTION EFFICIENCY USING SEXED BULL SPERM SORTED WITH TWO TYPES OF CELL SORTER

2011 ◽  
Vol 23 (1) ◽  
pp. 238
Author(s):  
H. Hayakawa ◽  
T.-I. Hirata
Keyword(s):  
Production Efficiency ◽  
Bos Taurus ◽  
Digital Processing ◽  
Embryo Production ◽  
Cell Sorter ◽  
Treatment Groups ◽  
Fort Collins ◽  
In Vitro Embryo Production ◽  
Bull Sperm

Cell sorting is an important part of the sperm sexing process. The objective of this study was to compare the efficiency of in vitro embryo production using sexed frozen–thawed bull sperm sorted with 2 types of cell sorter. Ejaculates from 2 Bos taurus (Holstein, 5 years old) bulls underwent conventional processing (control) or sorting for X chromosome bearing sperm using MoFlo® SX (SX, Dako, Fort Collins, CO, USA) or MoFlo® XDP-SX (XDP, Beckman Coulter, Fullerton, CA, USA) following XY™ sperm-sorting protocols. Processed sperm samples were cryopreserved in 0.5-mL plastic straws. Cumulus–oocyte complexes obtained from abattoir-derived ovaries were matured for 20 h in HEPES–TCM-199 (Lu and Seidel 2004 Theriogenology 62, 819–830) and randomly assigned to each of 3 sperm treatment groups. Thawed sperm were centrifuged for 20 min at 448 × g through an ISolate® (Irvine Scientific, Santa Ana, CA, USA) gradient (45:90%). Sperm pellets were washed in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) by centrifugation for 5 min at 252 × g. Oocytes were co-incubated with washed sperm (5 to 10 × 106 sperm mL–1) in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) for 8 h at 38.5°C in 5% CO2 and 95% air (Day 0). Presumptive zygotes were cultured for 90 h in CDM-1 (Lu and Seidel 2004 Theriogenology 62, 819–830) and then washed and cultured in IVD101 (Hoshi 2003 Theriogenology 59, 675–685) at 38.5°C in 5% CO2, 5% O2, and 90% N2. Cleavage rates on Day 2 and blastocyst rates on Day 7 to 9 were recorded after insemination. Two-way ANOVA was used for data analysis, followed by Fisher’s PLSD test. Experiments were replicated 4 times for bull A (total of 1 350 oocytes used) and 5 times for bull B (total of 1 529 oocytes used). The data are summarised in Table 1. No interaction was observed between the treatments and bulls. Cleavage rates were not significantly different in the 3 treatment groups. However, blastocyst rates were significantly lower in both SX (P < 0.001) and XDP (P < 0.002) groups than in control groups for both bulls but not different between SX and XDP (P > 0.8). Bull B showed significantly poorer results than bull A regarding both cleavage (P < 0.003) and blastocyst (P < 0.02) rates. MoFlo® SX (analogue processing) has been used for a decade, and XDP (digital processing) is the replacement model with its accelerated sorting speed. The current results indicated that the in vitro embryo production efficiency did not differ between sperm sorted with either SX or XDP. We suggest that sperm can be sorted using XDP without compromising sperm health. Table 1.Cleavage and blastocyst rates after IVF with 2 Holstein bulls for three sperm treatments

179 COMPARISON OF OOCYTE AND EMBRYO PRODUCTION AMONG BOS TAURUS, BOS INDICUS, AND INDICUS-TAURUS DONOR COWS

2010 ◽  
Vol 22 (1) ◽  
pp. 248 ◽  
Author(s):  
J. H. F. Pontes ◽  
K. C. F. Silva ◽  
A. C. Basso ◽  
C. R. Ferreira ◽  
G. M. G. Santos ◽  
...  
Keyword(s):  
High Efficiency ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Cumulus Cells ◽  
Holstein Cows ◽  
Embryo Production ◽  
Total N ◽  
The Mean ◽  
Donor Cows

In recent years, Brazil has become the leading country in the world for the number of embryos produced in vitro (Thibier M 2009 IETS Embryo Transfer Newsletter 22, 12-19). This is partly due to the large numbers of Bos indicus animals in Brazil, making up about 80% of the total cattle. The mean oocyte production per ultrasound-guided follicular aspiration from Bos indicus is higher than those for European breeds (Pontes JHF et al. 2009 Theriogenology 71, 690-697). In the present study, we analyzed 5407 ovum pick ups (OPU) and compared the average production of total (n = 90,086) and viable (n = 64,826) oocytes and the number of embryos produced in vitro from Gir (Bos taurus indicus), Holstein (Bos taurus taurus), 1/4 Holstein × 3/4 Gir, and 1/2 Holstein-Gir crossbreed cows. To obtain oocytes, OPU was repeated from 4 to 7 times (mean = 5.7 ± 2.4) in each donor cow aged from 3 to 7 years (mean = 5.0 ± 2.3) during a 12-mo period. COCs (n = 90,086) obtained were classified according to the presence of cumulus cells and the oocyte cytoplasm aspect (homogeneous or heterogeneous/fragmented). The viable oocytes (n = 64,826) were in vitro matured for 24 h at 38.8°C in an atmosphere of 5% CO2 in air. Since this was a commercial programm, frozen sexed semen (2 × 106 mL-1) from Gir (n = 8) or Holstein (n = 7) sires previously tested for high efficiency was used for IVF. Fertilization was carried out (18-20 h) and the presumed embryos were cultured for 7 days in the same conditions as were used for IVM. Data were analyzed by ANOVA. On average, 16.7 ± 6.2 oocytes were obtained per OPU/IVF procedure and 71.96% were considered viable. The mean numbers of total oocytes per OPU/IVF procedure were 17.1 ± 4.4 for Gir cows (n = 617), 11.4 ± 3.9 for Holstein cows (n = 180), 20.4 ± 5.8 for 1/4 Holstein × 3/4 Gir (n = 44), and 31.4 ± 5.6 for 1/2 Holstein-Gir crossbreed females (n = 37, P < 0.01). The mean numbers of viable oocytes per OPU/IVF procedure were 12.1 ± 3.8 for Gir cows, 8.0 ± 2.6 for Holstein cows, 16.8, ± 5.0 for 1/4 Holstein × 3/4 Gir, and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbreed females (P < 0.01). The average number of embryos produced by OPU/IVF were 3.2 (n = 12,243/3378) for Gir cows, 2.2 (n = 2426/1138) for Holstein cows, 3.9 (n = 1033/267) for 1/4 Holstein × 3/4 Gir, and 5.5 (n = 1222/224) for 1/2 Holstein-Gir. The average number of embryos produced per IVF session from 1/2 taurus × indicus donor cows was greater (P < 0.01) than from Bos indicus cows. The number of recoverable and viable oocytes and the number of embryos produced in vitro from Bos indicus donors were higher than from Bos taurus females. Therefore, the highest oocyte yield and the greatest embryo production were obtained from 1/2 taurus × indicus females. This work was supported by In Vitro Brasil.

In vitro Supplementation of Linoleic Acid Improves Quality of Cryopreserved Buffalo Semen

2020 ◽  
Author(s):  
R Ejaz ◽  
S Qadeer ◽  
M S Ansari ◽  
B A Rakha ◽  
S Shamas ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Membrane Integrity ◽  
Sperm Chromatin ◽  
Standard Procedures ◽  
Buffalo Semen ◽  
Chromatin Integrity ◽  
Live Sperm ◽  
And Control

Present study was designed to evaluate the effect of linoleic acid (LA) supplementation in extender on post thaw quality of cryopreserved buffalo semen. Semen was collected from three adult Nili Ravi buffalo bulls of same age with artificial vagina (42°C) for five weeks (replicates; N=30). Qualified semen ejaculates (>1mL volume, >60% motility, >0.5 billion/mL concentration) were diluted in tris-citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0ng mL-1 of LA and were cryopreserved using standard procedures. Sperm motility and plasma membrane integrity were improved plessthan0.05) in extender containing 10.0 ng mL-1 of LA compared to other treatments and control while number of acrosome intact live sperm, chromatin integrity and number of morphologically normal sperms remained the same. In conclusion, LA supplementation in extender at 10.0 ng mL-1 was found to be beneficial to improve post thaw quality of cryopreserved buffalo semen.

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