The goat is one of the most important domestic farm animals and provides a large number of products, such as milk, meat and hides. In vitro embryo production technology is an important tool for genetic improvement in goats and provides a source of low-cost embryos for basic research and for commercial application of biotechnologies, such as nuclear transfer, transgenesis, embryo sexing, and stem cells. In vitro goat embryo production includes three steps: in vitro maturation of goat oocytes, In Vitro Fertilization (IVF) of mature goat oocytes, and in vitro goat embryo culture
[1]
. IVF is a complex procedure whose success depends on several factors, such as the sperm concentration, fertilization media and IVF duration used.
The fertilization media used for IVF are the Synthetic Oviductal Fluid (SOF) medium used in ovine
[2]
, Brackett and Oliphant (BO) medium used in caprine
[3]
, and the Tyrode’s Albumin Lactate Pyruvate (TALP) medium supplemented with hypotaurine widely used in goat
[4]
. For IVF, the final sperm concentration used in the IVF drop can vary from 0.5 × 106 cells/ml to 12 - 15 × 106 cells/ml, depending on the IVF used
[5]
[6]
. According to Souza-Fabjan et al. (2023)
[1]
, co-culture time is too short or long can reduce the IVF rate. These authors showed that a shorter oocyte-sperm incubation reduced IVF efficiency, but using longer co-culture time could result in higher ROS concentrations and lower blastocyst rate. In IVF goats, the duration of sperm and oocyte co-culture is inconsistent between the studies. Sperm and oocytes of goats are co-cultured for 16 to 24 hours, depending on the laboratory.
Cỏ goat is an indigenous goat breed, and they are an important genetic resource for conservation of native Vietnamese goat biodiversity. Cỏ goats are a goat breed commonly raised in Vietnam for meat production. Some of the advantages of Cỏ goats include small in size and popular for disease resistance, high reproduction, limited food requirement and high adaptability in Vietnam’s climate
[7]
. The development of in vitro production of goat embryos is able to support the propagation of this animal, however, IVF experiments on Cỏ goat in Vietnam have not been done in the past. Therefore, in this study, we optimized the in vitro fertilization system in Cỏ goat oocytes.
2. Materials and Methods
All the experimental procedures used in this study were performed in accordance with Vietnam legislation and according to Decision No. 5814/QĐ-BNN-KHCN of the Ministry of Agriculture and Rural Development in Vietnam on December 27.2023.
2.1. Reagents and Chemicals
All reagents and chemicals used in this study were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Petri dishes used for cell culture are originated from Corning Inc. (Corning, NY, USA).
2.2. Collection of Cỏ Goat Ovaries and Cumulus Oocytes Complexes (COCs)
Cỏ goat ovaries were transported to the laboratory in Dullbeco Phosphate Buffer Saline (DPBS) supplemented with antibiotics within 2 hours after collection from the slaughterhouse. Oocytes were collected from 2 - 8 mm diameter follicles on the ovarian surface using an aspiration method with a 5 ml syringe containing Tyrode’s Albumin Lactate Pyruvate-HEPES (TALP-HEPES) oocyte collection solution supplemented with serum and an 18G needle. After aspiration, the TALP-HEPES medium with harvested oocytes was transferred to a Petri dish, and oocytes were searched by using a stereo microscope and evaluated according to the standards of Wani et al. (2000)
[8]
. After evaluation, cumulus oocytes complexes were selected based on: 1) uniform cytoplasm and 2) the presence of at least three compact surrounding layers of cumulus cells.
2.3. In Vitro Maturation of Goat Oocytes
The COCs of Cỏ goat oocytes were washed three times in IVM medium either TCM 199 supplemented with 10% Fetal Cafl Serum (FCS), 50 ng/ml Follicle Stimulating Hormone (FSH), 10 ng/ml Epidermal Growth Factor (EGF), 100 µM cysteamine, 100 units/ml penicillin G potassium + 0.1 mg/ml streptomycin sulphate, and then transferred to 4-well plates containing 500 µl of the in vitro maturation medium per well for 22 hours, under conditions of 38.5˚C, 5% CO2, and saturated air humidity (50 oocytes per well).
2.4. In Vitro Fertilization (IVF) and Embryo Culture (IVC)
The IVF and IVC procedures were performed using the method of Widayati and Pangestu (2020)
[9]
with some modifications. Matured Cỏ goat oocytes were washed in in vitro fertilization (BO-IVF) medium two times. Thawed semen was centrifuged at 320 × g for 5 min in BO sperm washing medium. The supernatant was removed, and sperm pellet was diluted in BO sperm washing medium. Oocytes and sperm were incubated in IVF medium from 18 to 24 h depending on the experiment, at 38.5˚C under 5% CO2, 5% O2 and humidified air. After duration co-culture, the cumulus cells surrounding the oocyte were removed by repeated pipetting in TALP-HEPES medium + 0.2% hyaluronidase. The oocytes without cumulus cells were washed in SOF medium two times and cultured in SOF medium + 2.5% Fetal Bovine Serum (FBS) in an incubator at 38.5˚C under 5% CO2, 5% O2 and humidified air for 7 days.
2.5. Evaluation of Embryo Cell Number
The cell numbers in blastocyst at Day 7 after IVF were evaluated by staining with Hoechst 33342. The embryos of Cỏ goat were washed in Phosphate Buffered Saline (PBS) medium supplemented with 0.3% Polyvinylpyrrolidone (PVP) solution. Next, the oocytes were transferred into a staining solution (Hoechst 33342 + Absolute ethanol in a 1:9 ratio) and left overnight at 4˚C. After incubation overnight at 4˚C in the staining solution, the embryos were washed in absolute ethanol and then transferred to a Glycerol solution. Subsequently, the embryos were transferred to a glass slide, each embryo in a drop, and aligned along the length of the slide. A cover slip was placed over the slide, and the embryos were examined under a fluorescence microscope. The cell numbers count under a fluorescence microscope.
Experiment 1. The effect of IVF media on the in vitro fertilization of Cỏ goat oocytes
In this experiment, matured Cỏ goat oocytes were divided into two IVF media: 1) TALP-Fert and 2) BO-IVF medium. Matured Cỏ goat oocytes and sperm were co-cultured for 20 hours and placed in an incubator at 38.5˚C, in 5% CO2, 5% O2 and in saturated air humidity. The embryo developmental competence of Cỏ goat oocytes was evaluated by examination of cleavage, blastocyst and hatching rates at Day 2, Day 6, and Day 7 after IVF, respectively, and total cells per blastocyst counted on Day 7. Eight replications were performed.
Experiment 2. The effect of sperm concentration on the in vitro fertilization of Cỏ goat oocytes
In this experiment, matured Cỏ goat oocytes were fertilized in the selected fertilization medium from Experiment 1 for four sperm concentrations: 5 × 105, 1 × 106, 2 × 106 and 3 × 106 sperm/ml. Cỏ goat oocytes and sperm were co-cultured for 20 hours and placed in an incubator at 38.5˚C, in 5% CO2, 5% O2 and in saturated air humidity. The IVF of matured Cỏ goat oocytes was performed as described above. The embryo developmental competence of Cỏ goat oocytes was evaluated by examination of cleavage, blastocyst and hatching rates at Day 2, Day 6, and Day 7 after IVF, respectively, and total cells per blastocyst counted on Day 7. Eight replications were performed.
Figure 1. 2 - 4 cells embryo of Cỏ goat at Day 2 after IVF (eyepiece * objective is 5×/0.12).
Figure 2. Blastocyst and hatching blastocyst of Cỏ goat at Day 7 after IVF (eyepiece * objective is 10×/0.25).
Experiment 3. The effect of IVF duration on the in vitro fertilization of Cỏ goat oocytes
In this experiment, Cỏ goat oocytes were fertilized in the selected fertilization medium, sperm concentration from Experiment 1 and Experiment 2 for 18, 20, 22 and 24 hours at 38.5˚C, in 5% CO2, and in saturated air humidity. The IVF of Cỏ goat oocytes was performed as described above. The embryo developmental competence of Cỏ goat oocytes was evaluated by examination of cleavage, blastocyst and hatching rates at Day 2, Day 6, and Day 7 after IVF, respectively, and total cells per blastocyst counted on Day 7. Eight replications were performed.
2.7. Statistical Analysis
All data were expressed as mean ± SEM values and analysed by ANOVA, followed by Tukey’s multiple comparisons test, using GraphPad Prism software (Version 7.02 for Windows, GraphPad Software, La Jolla, California, USA. P < 0.05 was defined as the significance level.
3. Results3.1. Experiment 1. The Effect of IVF Media on the in Vitro Fertilization of Cỏ Goat Oocytes
The results in
Table 1
indicated no significant difference in the cleavage and blastocyst and hatching rates and the average number of cells/blastocyst of Cỏ goat oocytes after fertilization between two IVF media: TALP-Fert and BO-IVF (67.84% vs. 68.92%, 23.82% vs. 24.76%, 4.36% vs. 4.02% and 132.96 vs. 135.08, respectively; P > 0.05).
<xref ref-type="bibr" rid="scirp.138470-"></xref>Table 1. Influence of IVF media on the in vitro fertilization of Cỏ goat oocytes.
Medium
No. of fertilized oocytes
No. of cleavage
(% total)
No. of blastocyst
(% total)
No. of hatching blastocyst
(% total)
The average number of cells/blastocyst
TALP-Fert
98
66
67.84 ± 2.34
23
23.82 ± 2.15
4
4.36 ± 1.98
132.96 ± 2.58
BO-IVF
102
70
68.92 ± 2.22
25
24.76 ± 2.05
4
4.02 ± 2.18
135.08 ± 2.21
Eight replications were performed. Percentage data are shown as mean ± SEM.
3.2. Experiment 2. The Effect of Sperm Concentration on the in Vitro Fertilization of Cỏ Goat Oocytes
Based on the results of Experiment 1, in Experiment 2 we used the BO-IVF medium to evaluate the effect of sperm concentration on the in vitro fertilization of Cỏ goat oocytes. The results are shown in
Table 2
. The cleavage rate of 5 × 105 group was lower than that of 1 × 106, 2 × 106 and 3 × 106 groups (55.96% vs. 69.98%, 76.48%, 76.95%; respectively, P < 0.05). The blastocyst rate of 2 × 106 and 3 × 106 groups was higher than that of 5 × 105 and 1 × 106 groups (35.92% and 35.96% vs. 14.72% and 24.01%; respectively, P < 0.05). The difference in blastocyst and hatching blastocyst rates between 2 × 106 and 3 × 106 groups were not statistically significant (P > 0.05).
<xref ref-type="bibr" rid="scirp.138470-"></xref>Table 2. Influence of sperm concentration on the in vitro fertilization of Cỏ goat oocytes.
Concentration
(sperm/ml)
No. of fertilized oocytes
No. of cleavage
(% total)
No. of blastocyst
(% total)
No. of hatching blastocyst
(% total)
The average number of cells/blastocyst
5 × 105
114
63
55.96a ± 2.07
16
14.72a ± 2.31
3
2.96 ± 1.75
133.24 ± 2.24
1 × 106
119
83
69.98b ± 2.03
28
24.01b ± 2.42
4
3.89 ± 2.76
134.98 ± 2.38
2 × 106
118
90
76.48b ± 1.99
42
35.92b ± 2.14
6
5.48 ± 2.35
135.01 ± 2.46
3 × 106
112
86
76.95b ± 2.58
40
35.96b ± 2.67
6
5.62 ± 2.32
135.78 ± 2.51
Eight replications were performed. Percentage data are shown as mean ± SEM. a and b in the same column differ significantly (P < 0.05).
3.3. Experiment 3. The Effect of IVF Duration on the in Vitro Fertilization of Cỏ Goat Oocytes
Based on the results of Experiment 1 and Experiment 2, in Experiment 3, matured Cỏ goat oocytes were fertilized in the BO-IVF medium with a concentration of 3 × 106 sperm/ml in each IVF droplet.
The results in
Table 3
indicate that the cleavage, blastocyst, and hatching blastocyst rates of 18 hours group were lower than that of 20-, 22- and 24-hour groups (54.16% vs. 76.22%, 77.21% and 77.09%; 17.64% vs. 35.67%, 35.82% and 35.96%; 3.58% vs. 5.92%, 6.36, 6.31%; respectively, P < 0.05). The difference in cleavage, blastocyst and hatching blastocyst rates between 20-, 22- and 24-hour groups were not statistically significant (P > 0.05).
<xref ref-type="bibr" rid="scirp.138470-"></xref>Table 3. Influence of IVF duration on the in vitro fertilization of Cỏ goat oocytes.
IVF duration
(hours)
No. of fertilized oocytes
No. of cleavage
(% total)
No. of blastocyst
(% total)
No. of hatching blastocyst
(% total)
The average number of cells/blastocyst
18
115
62
54.16a ± 2.31
20
17.64a ± 2.06
4
3.58 ± 2.33
132.98 ± 2.46
20
121
92
76.22b ± 2.26
43
35.67b ± 2.41
7
5.92 ± 2.09
134.89 ± 2.14
22
117
90
77.21b ± 2.54
41
35.82b ± 2.28
7
6.36 ± 2.47
135.12 ± 2.71
24
116
89
77.09b ± 2.72
41
35.96b ± 2.45
7
6.31 ± 2.39
134.86 ± 2.67
Eight replications were performed. Percentage data are shown as mean ± SEM. a and b in the same column differ significantly (P < 0.05).
4. Discussion
The results of this study indicate that IVF media does not affect on the in vitro fertilization of Cỏ goat. There was no significant difference in the cleavage and blastocyst and hatching rates and the average number of cells/blastocyst of Cỏ goat oocytes after fertilization between TALP-Fert and BO-IVF media (
Table 1
, P > 0.05). The quality of sperm has an important role in the success of the IVF process and in vitro embryo production. During the IVF process, there are some changes in membrane properties, intracellular ion concentrations, enzyme activity, protein modifications that can affect the IVF process
[10]
. To improve fertilization ability and capacitation of sperm as well as increase the number of fertilized oocytes, adding of supplements to the IVF media is necessary. According to Kharche et al. (2009)
[11]
, when using fetal calf serum instead of bovine serum albumin in IVF media will increase the cleavage and blastocyst rates of goat oocytes. Heparin and hypotaurine are agents to improve the ability of sperm fertilization in vitro
[12]
[13]
.
In this study, Heparin was added to the BO-IVF media to improve the effect of IVF of goat oocytes. Cox and Alfaro (2007)
[14]
also showed that heparin is a potent glycosaminoglycan that has been shown to improve the fertilizing ability of bovine, goat and sheep when used in IVF medium. The cause is that the presence of heparin in the IVF media will induce capacitation of sperm in the in vitro embryo production, increased calcium uptake, increased intracellular free calcium and decreased calmodulin concentration in spermatozoa
[15]
.
Hypotaurine is also an antioxidant, which can induce capacitation of sperm during IVF process, and it is often oxidized to taurine to remove hydroxyl radicals in the in vitro fertilization system
[16]
. According to Pons-Rejraji et al. (2021)
[17]
, the presence of heparin and hypotaurine in the IVF media will increase viability and progressive motility, as well as reduce the rate of sperm that undergo chromatin decondensation, DNA fragmentation and nuclear vacuolization, which is why in our study, we added Hypotaurine to TALP-Fert medium and heparine to BO-IVF medium.
In Experiment 2, our results are similar to Palomo et al. (2010)
[18]
. According to Palomo et al. (2010)
[18]
, sperm concentration affects the in vitro fertilization of prepubertal goat oocytes. However, there is no consensus among studies on the optimal of sperm concentration for IVF in goat. Palomo et al. (2010)
[18]
reported the optimum sperm concentration is 4 × 106 sperm/ml, but in our study and the report of Pawshe et al. (1994)
[19]
, the optimum sperm concentration is 2 × 106 or 3 × 106 sperm/ml. The reason for these different results might be the different evaluation criteria, the quality of sperm between the studies. For optimum concentration sperm, Palomo et al. (2010)
[18]
used the penetration rate as evaluation criteria, whereas we and Pawshe et al. (1994)
[19]
used cleavage and blastocyst and hatching blastocyst rates.
The correlation between the polyspermy rate and sperm concentration present during IVF process show that reducing sperm concentration may be minimize polyspermy in IVF (Gil et al. 2008)
[20]
. The quality of sperm frozen-thawed is a necessary requirement to increase the fertilized oocytes rate. During in vitro fertilization, when using sperm frozen, high sperm concentration is essential to maintain the motility of sperm and increase the number of sperm capable of fertilization. Therefore, this could be the reason why the optimal of sperm concentration in this study was lower than that of the report of Palomo et al. (2010)
[18]
. When increasing sperm concentration from 5 × 106 to 10 × 106 sperm/ml, the fertilized oocytes rate decreases and the polyspermy rate increase. Therefore, in this study, we also limited sperm concentration to a maximum of 3 × 106 sperm/ml.
In this study, we aimed to optimize in vitro fertilization for Cỏ goat oocytes in order to optimize the production of blastocyst goat embryo by IVF. Optimization of the IVF duration means the maximization of efficacy as measured by blastocyst stage embryos after subsequent IVF. The results of Experiment 3 showed that IVF duration affect the in vitro fertilization of Cỏ goat. Our results are similar to Cognié et al. (2003)
[21]
and Tan et al. (2011)
[22]
. According to Cognié et al. (2003)
[21]
and Tan et al. (2011)
[22]
, IVF duration affect the in vitro fertilization of goat oocytes. The period of sperm-oocytes exposure in goat is still controversial, and there is no consensus on the optimal IVF duration in goat between studies: 20 h
[21]
, 24 h
[23]
[24]
, 16 - 20 h
[14]
, 18 h
[3]
and 12 h
[25]
. Tan et al. (2011)
[22]
showed that the cleavage rate of 8 - 14 h group was higher than the 18 - 24 h group, whereas in the report of Cognié et al. (2003)
[21]
, the cleavage and blastocyst rates of 17 h group were higher than the 3 h group. Differences between studies may be due to the quality of sperm and matured oocytes, IVF medium, IVF protocol…
According to Van Soom et al. (2002)
[26]
, the presence of cumulus cells around the oocytes at the time of fertilization appears to increase the blastocyst yield. dos Santos-Neto et al. (2020)
[27]
found beneficial effects of cumulus cells during in vitro fertilization in goat. The cumulus cells act as a physical barrier in polyspermy control, and they may exert an additional benefit in the gamete interaction. Even, Yanagimachi (2022)
[28]
suggested that progesterone secreted by cumulus cells can activate the acrosome reaction of sperm during IVF. That is why, in this study to increase the rate of Cỏ goat blastocyst, we used the IVF process with cumulus cells in Cỏ goat oocytes.
According to Mauchart et al. (2023)
[29]
, increasing the co-incubator time of sperm oocytes can also cause cytotoxic damage due to increased amount of reactive oxygen species (ROS). Similarly, Gualtieri et al. (2021)
[30]
also found that using frozen sperm during IVF process produced increased amounts of ROS. ROS are by-products of metabolism and cell death, which may be related to longer fertilization duration. ROS has played an important role as it increases DNA fragmentation, modifies the cytoskeleton and produces a loss of fluidity, integrity and competence of the sperm membrane to participate in the membrane events associated with fertilization
[30]
. Fan et al. (2023)
[31]
showed that reducing the co-incubator time of sperm oocytes improves the viability of human embryo, possibly due to a decrease in potential damage from sperm metabolic waste products. Therefore, determining the optimal co-incubator time of sperm oocytes will improve the efficiency of in vitro goat embryo production.
5. Conclusion
In conclusion, the present study reveals that the matured Cỏ goat oocytes were fertilized in BO-IVF medium with sperm concentration of 3 × 106 sperm/ml for 20 h, which is suitable for the in vitro Cỏ goat embryo production.
Acknowledgements
The research was carried out at the Key Laboratory of Animal Cell Technology. The authors would like to thank the Vietnamese Ministry of Science and Technology for supporting the project.
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