Description of the processes to obtain bovine
1. Introduction
Interferon-tau (IFN-τ) is secreted by the mononuclear cells of the primitive extra-embryonic trophoblast (Farin et al. 1989; Guillomot et al. 1990) which will become the main part of the future placenta, in ruminants. This cytokine is constitutively produced during the short period of the conceptus periimplantation (Martal et al. 1979; Godkin et al. 1982; Hansen et al. 1988; Charlier et al. 1989). It plays an essential role for maternal recognition of pregnancy, particularly allowing the maintenance of the
The regulation of IFN-τ secretion remains poorly understood (Martal et al. 1998; Yamagushi et al. 2001; Demmers et al. 2001; Ezashi et al. 2001; Stewart et al. 2002; Spencer and Bazer 2002). Some growth factors and cytokines are implicated in the positive control of IFN-τ secretion such asInsulin-like Growth Factor (IGF-I ) and IGF-II (Ko et al. 1991), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukine-3 (Imakawa et al. 1993, 1995; Emond et al. 2004). Indeed, these factors are secreted by trophoblast and endometrium in early pregnancy (Mathialagan et al. 1992; Vogiagis et al. 1997; Martal et al. 1997, 2002). The induction of IFN-τ expression appeared genetically determined because IFN-τ secretion has been evidenced from hatched blastocysts completely produced
Generally, the evaluation of the development potential of embryos today still depends on subjective morphological examination (Lindner and Wright, 1983; Buttler and Biggers 1989; Shamsuddin et al. 1992, Massip et al. 1995). However, the development stage and quality of the embryos significantly influence the IFN-τ secretion. Some authors have proposed that the produced amount of IFN-τ could be a useful objective indicator of embryo quality (Hernandez-Ledezma et al. 1992, 1993). But others, considering that the age of blastocysts formation
The aim of this study was therefore to compare the IFN-τ secretion after hatching in bovine blastocysts of good homogeneous quality wether they are produced
2. Materials and methods
2.1. Production of in vitro bovine blastocysts
2.1.1. In vitro maturation of oocytes
Unless otherwise indicated, all chemicals in this study were purchased from Sigma-Aldrich, (Saint Quentin. Fallavier, France). Ovaries from Prim Holstein cows were quickly collected after death in a local slaughter-house and transferred to the laboratory into a saline solution with 0.9% (w/v) NaCl at approximately 35°C. The largest interval between animal killing and oocyte conditioning was 3 hours. Cumulus-oocyte complexes (COCs) were recovered by aspiration of follicles of 2-8 mm in diameter using a 18 gauge needle under vacuum pressure of approximately 50 mm Hg. The COCs were collected into Hepes-buffered tissue culture medium 199 (TCM 199 ref. M-7528) supplemented with 0.4% (w/v) BSA (Cohn Fraction V, ref. 9647). Before
2.1.2. In vitro fertilization
2.1.3. In vitro culture
After 20 hours, the
2.2. Production of in vivo bovine blastocysts
The oestrous cycles of french Prim Holstein donor cows were synchronised with an implant of 3mg Norgestomet-3.8 mg valerate oestradiol (Crestar®INTERVET S.A.- Holland) for 9 days. The oestrous reference was observed 48-68 hours after removing the implant. The cows were superovulated 8 to 12 days after oestrous with p-FSH (Stimufol®Merial SAS - France). A total dose of 450µg p-FSH was administered (8 i.m. injections with decreasing doses for 4 days) and an analogue of PGF2α (500 µg i.m.; Cloprostenol Estrumate® Schering Plough Vet. S.A.) was injected at the moment of the 5th p-FSH injection. Two artificial inseminations (IA) were performed 12 and 24 hours after starting oestrous (day 0 ). The frozen-thawed semen for IA of embryos donor cows was the same that used for the production of
2.3. Determination of IFN-τantiviral activity
Embryo culture media were assayed for antiviral activity by use of a cytopathic effect titration assay that used a bovine kidney cell line designated Madin and Darby Bovine Kidney (MDBK cells) challenged with vesicular stomatitis virus. The extent of cell protection against viral lysis was compared with that of a diluted human INF-α (Alpha therapeutics Corporation Los Angeles, CA) used as a standard. This standard possesses 8.8 International Reference Units of activity per laboratory unit (u) when compared to human INF-α (Leukocyte IFN standard: GA-23-902-530 reference sample, obtained from NIH Bethesda MD, USA.)
The sensitivity of the IFN-τassay was 0.012 u /100µl. Titrated IFN were expressed in laboratory units/embryo/24h, where one unit was equivalent to that amount of IFN that protected 50% of the MDBK cells monolayer from lysis upon exposure to the cytopathic effects of the virus. Immunoneutralization assays were performed with polyclonal antisera raised against IFN-τ. Briefly, IFN samples were serially diluted in a given dilution of anti-INF antisera (L’Haridon 1991). The excess of immunoneutralization results was determined from the IFN-τactivity compared with that of untreated controls.
2.4. Determination of the numbers of inner mass (ICM) and trophectoderm (TE) cells of blastocysts produced in vitro and ex vivo.
Equal numbers of hatched blastocysts (per treatment group, completely
2.5. Experimental design
The aim of this experiment was to compare IFN-τ production of blastocysts
2.6. Statistical analysis
Data on embryos origin (
3.Results
3.1. Obtaining homogeneous expanded blastocysts of in vitro and in vivo origins on day 7
The results of the processes to obtain derived
|
Oocytes n |
Cleavage day 2 % (n) |
Day 7 | Day 8 | Total % (n) | |||||||||
Blastocysts % (n) |
Expanded Blastocysts % (n) |
Total % (n) |
|
Blastocysts % (n) |
Expanded Blastocysts % (n) |
Total % (n) |
||||||||
559 |
83 (464) |
17 (98) |
8 (44) |
25 (142) |
|
6 (32) |
17 (94) |
23 (126) |
48 (268) |
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|
Superovulated cows n |
Recovered embryos % (n) |
Recovered embryos on day 7 | |||||||||||
Morulas % (n) |
Blastocysts % (n) |
Expanded Blastocysts % (n) |
Degenerated Embryos % (n) |
|
||||||||||
11 |
100 (87) |
18 (16) |
28 (24) |
32 (28) |
22 (I9) |
|
3.2. Effect of embryos origin on the IFN-τ production (Table 2).
On day 8, after 24h-culture,96% blastocysts (n=46) completely produced
On day 10, after 72 h-culture 63% group A (n=29) exhibits an average IFN-τ production of 491 ± 128 pM versus 216 ± 37 pM for group B (58%, n=23) (NS). The rest of group A (19% n=9) and those of group B (13%, n=5) both secreted <54 pM IFN-τ. After 96 h-culture 57% group A (n= 26) had an average IFN-τ production of 499 ± 135 pM versus 353 ± 93 pM for group B (53%, n=21) (NS). The rest of group A (6% n=3) and those of group B (8%, n=3) both produced <54 pM IFN-τ.
Time culture (hours) |
|
|
||
% (n) | IFN-τ concentration mean ± sem (pM) | % (n) | IFN-τ concentration mean ± sem (pM) | |
24 | 96(44) 4 (2) |
< 54 93 ± 0 |
100 (40) | <54 |
48 | 41 (19) 57 (26) |
143 ± 24 b < 54 |
53 (21) 47(19) |
85 ± 12 a < 54 |
72 | 63 (29) 19 (9) |
491 ± 128 a <54 |
58 (23) 13 (5) |
216 ± 37 a < 54 |
96 | 57 (26) 6 (3) |
499 ± 135 a < 54 |
53 (21) 8 (3) |
353 ± 93 a < 54 |
120 | 48 (22) 13 (6) |
559 ± 136 a < 54 |
50 (20) 10 (4) |
333 ± 75 a < 54 |
The average of accumulated IFN-τ production per embryo from days 7 to 10 ( 72 hours) of group A was 550±129 pM IFN-τ (n=38) versus group B 277±41pM IFN-τ (n=28) (p<0.05) figure 1. At this stage of culture, on day 10 after fertilization, the morphological aspects of completely produced
Between days 7 to 11 (96 hours), the production of IFN-τ increased in 1100±20 pM IFN-τ (n=29) for group A versus 670±117 pM IFN-τ (n=24) for group B, (p<0.05). Between days 7 to 12 (120 hours) of
According to the linear regression analysis, the IFN-τ production increases during the time of culture independently of the embryos origins (r = 0.13 for group A, r = 0.23 for group B; p > 0.05).
3.3. Relationship between embryo quality and accumulated IFN-τ production by embryo, independently of its origin
The average IFN-τ production from days 7 to 10 (72h-culture) in relationship with embryo quality did not show significant differences, (p>0.05): for quality I (excellent) the average IFN-τ production was 534±138 pM IFN-τ (n=21), for quality I (good) 389±101 pM IFN-τ (n=29) and for quality II (fair) 328±108 pM (n=5). The average IFN-τ production from days 7 to 11 (96h-culture) in relationship with embryo quality did not show significant differences (p>0.05); 1040±216 pM IFN-τ (n=17); 870±158 pM (n=33) and 507±262 pM (n=5),respectively. But the average IFN-τ production from days 7 to 12 (120h-culture) in relationship with embryo quality shows significant differences between excellent quality (1815±453 pM, n=10) or good quality (1356±200 pM, n=29) with fair quality (360±188 pM, n=4), (p<0.02), Table 3.
The logistic regression analysis of the relationship between the embryo quality and IFN-τ production showed predicted probabilities from observed responses: Concordance: 60.3% and Discordance: 23.9%.
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n (%) |
n (%) |
IFN-τ concentration mean ± sem (pM) | n (%) |
IFN-τ concentration mean ± sem (pM) | n (%) |
IFN-τ concentration mean ± sem (pM) | |
Q1(Ex.) | 81 (70) |
24 (21) |
534 ±138a | 20 (17) |
1040 ± 216a | 10 (12) |
1815 ± 453 a |
Q1(G.) | 19 (16) |
34 (29) |
389 ±101a | 38 (33) |
870 ± 158a | 29 (34) |
1356 ± 200ab |
Q II | - | 6 (5) |
328 ± 108a | 6 (5) |
507± 262a | 4 (5) |
360 ± 188 b |
4. Discussion and conclusion
Our average results for producing bovine blastocysts
The IFN-τ production on day 8, after 24 hours of
To compare well the IFN-τ production of
However, their early-forming blastocysts were generally considered more developmentally competent than those which formed late, and these last authors suggested a possible negative relationship between early IFN-τ production and competence. In fact, this hypothesis has not been verified in the present study, possibly because of the homogeneity of the embryos chosen on the good appearance on day 7 according to the usual morphological criteria. Embryo quality is usually based on series of subjective visual assessments of morphologic parameters evaluation which may include embryo shape, size, cellular integrity, appearance of the cytoplasm and nucleus and other often intangible criteria (Lindner and Wright, 1983; Shamsuddin et al. 1992, Massip et al. 1995). In this study, the embryos which maintained in good and excellent quality had a better production of IFN-τ compared with those which turned to fair quality. This can be explained by a higher number of degenerating cells; in others the present study shows that the embryo quality is associated with IFN-τ production and confirms earlier studies (Kubish et al. 1998; Hernández-Ledezma et al; 1993; Stojkovic et al. 1995 and 1999).
Finally this study leads to the conclusion that significant differences in the production of IFN-τ were found in relationship with embryo origin,
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