The effect of in vitro treatment of bovine embryos with IGF-1 on subsequent development in utero to Day 14 of gestation

Culture of bovine embryos with insulin-like growth factor-1 (IGF-1) can improve development to the blastocyst stage and embryo survival following transfer to heat-stressed, lactating dairy cows. Two experiments were conducted to determine whether IGF-1 could improve embryo survival and development at Day 14 after ovulation. In Experiment 1, non-lactating Holstein cows (n=58) were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced in vitro and cultured with or without 100ng/mL IGF-1. At Day 7 after expected ovulation (Day 0), groups of 7-12 embryos were randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and the presence or absence of an embryonic disc was recorded. Recovered embryos were cultured individually for 24h to determine interferon-tau (IFN-tau) secretion. There was no effect of IGF-1 on embryo recovery rate, embryo length or IFN-tau secretion. In Experiment 2, non-lactating (n=56) and lactating (n=35) Holstein cows were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced as described in Experiment 1. At Day 7 after expected ovulation (Day 0), a single embryo was randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and IFN-tau secretion were determined as in Experiment 1. Recovery rate at Day 14 tended (P=0.1) to be higher for recipients that received IGF-1 treated embryos compared to control embryos (43.2% versus 26.1%, respectively). There was no effect of IGF-1 on embryo length or IFN-tau secretion. In conclusion, results suggest that exposure to IGF-1 through Days 7-8 of development does not enhance capacity of embryos to prevent luteolysis. Results of the single embryo-transfer experiment suggested that IGF-1 treatment might affect embryo survival post-transfer as early as Day 14 after ovulation. Further experimentation is warranted to verify this finding.     

Heterologous anti-progesterone monoclonal antibody arrests early embryonic development and implantation in the ferret (Mustela putorius)

Early embryo development and implantation were arrested in ferrets passively immunized with a mouse monoclonal anti-progesterone antibody injected intraperitoneally at 72 and 96 h post coitum (p.c.) or at 72 h p.c. only. In control ferrets injected with mouse serum or 0.9% NaCl, implantation sites were found in all mated females; autopsies were carried out at Day 14 p.c. A total of 34 unimplanted embryos were recovered from the reproductive tract of antibody-treated ferrets and none of these had progressed to the blastocyst stage. When ferrets were treated with antibody at 72 h p.c. and autopsies were carried out at Day 6 p.c., only 1 of 29 embryos recovered had progressed beyond the 4-cell stage in 4 females. In 4 control animals most embryos recovered at Day 6 were at the morula (32%) or blastocyst (28%) stage. Embryos from ferrets treated with antibody were therefore developmentally arrested when recovered 72 h after antibody administration. Plasma progesterone concentrations were approximately 6-fold higher in antibody-treated ferrets with unimplanted embryos (711 +/- 132 nmol/l; 223 ng/ml) compared with control pregnant females (102 +/- 4 nmol/l; 32 ng/ml) at Day 14 p.c. The results are consistent with the hypothesis that the normal course of pregnancy is arrested as a result of antibody binding of progesterone in the circulation, presumably causing a decrease in the amount of progesterone available to target cell receptors, and that heterologous anti-progesterone antibody blocks normal cleavage and embryonic development at an early stage before cavitation.     

Status of oocytes and embryos of X-autosome translocation carrier sows

The impact of an X-autosome translocation t(Xp+; 14q-), on ovulation, fertilization and embryo survival in carrier sows, was examined and compared with these parameters of normal sows. Corpora lutea counts during week-2 and week-4 of gestation were similar in normal and carrier sows (14.4 +/- 1.36 and 15.5 +/- 2.18) although embryo recovery (11.0 +/- 1.87 and 6.0 +/- 1.47) was lower than that from normal sows (12.8 +/- 1.46 and 11.5 +/- 0.87), at these stages. Among the embryos karyotyped from the week-2 embryos of carrier sows, 42% were normal, 26.4% were carriers and 31.6% were of unbalanced chromosome make-up, and of the week-4 embryos of carriers, 33.3% were normal, 57.1% were carriers and 9.1% were chromosomally unbalanced females. The preponderance of females among the unbalanced embryos recovered at week-2 of gestation (11_ and 1_) and the total absence of males among those recovered at week-4, suggest that oocytes with unbalanced chromosome constitution are eliminated before week-2 of gestation if they are fertilized by Y bearing sperm, and that the unbalanced oocytes fertilized by X bearing sperm survive up to the peri-attachment stage even though all chromosomally unbalanced embryos are eliminated before term regardless of their sex.     

Ovulation, fertilization and early embryonic development in the bitch (Canis familiaris)

Using circulating plasma hormone estimations, ovulation was monitored in bitches. The results obtained indicate that the timing of ovulation bears little relationship to alterations in sexual behaviour. The bitches were killed and reproductive tracts were removed at various intervals after ovulation and ova or embryos were recovered. The embryo stages were assessed visually and some were investigated histologically. Embryonic development, to early blastocyst stage, took place within the oviducts during the first 12 days after ovulation and there was a marked increase in size between the early and late blastocyst. A culture system using cells from the uterine tube supported the development of one 1-cell embryo to the morula stage.     

Embryo survival and conceptus growth after reciprocal embryo transfer between Chinese Meishan and Landrace x Large White gilts

Embryos were transferred between Meishan and Landrace x Large White (control) gilts on Day 4 or 5 to establish approximately equal numbers of all four possible combinations of donor breed and recipient breed. The breed of the donor gilt significantly (P less than 0.01) affected embryo survival with 44.5% of transferred Meishan embryos and 69.6% of transferred control embryos surviving to Day 30 +/- 1. There was no influence of the breed of the recipient gilt on the proportion of embryos which survived. These differences in embryo survival between the two breeds could not be explained by differences in (1) the number of embryos transferred, (2) the stage of development of the embryos transferred, (3) the interval between ovulation and transfer or (4) the degree of asynchrony between donor and recipient gilt. On Day 30 +/- 1 embryos from control donors developed into longer fetuses (P less than 0.01) with larger allantoic sacs (P less than 0.05) than did embryos from Meishan donors. Fetuses in control recipients were longer (P less than 0.01), heavier (P less than 0.001) and had larger allantoic sacs (P less than 0.05) than fetuses occupying Meishan uteri. The interaction between breed of donor gilt and breed of recipient gilt did not significantly affect conceptus growth. These results suggest that Meishan pig embryos may be less tolerant to routine embryo transfer procedures than those of control gilts, that the genotype of the dam does not affect the proportion of embryos surviving to Day 30 +/- 1, and that both fetal and maternal factors affect conceptus growth.     

Prolonged mating in prairie voles (Microtus ochrogaster) increases likelihood of ovulation and embryo number

Prairie voles are induced ovulators that mate frequently in brief bouts over a period of approximately 24 h. We examined 1) impact of mating duration on ovulation and embryo number, 2) incidence of fertilization, 3) temporal pattern of embryo development, 4) embryo progression through the reproductive tract over time, and 5) embryo development in culture. Mating was videotaped to determine first copulation, and the ovaries were examined and the reproductive tracts flushed at 6, 8, 10, 12, 16, 20, and 24 h and 2, 3, and 4 days after first copulation. The number of mature follicles and fresh corpora lutea and the number and developmental stage of embryos were quantified. One, two-, and four-cell embryos were cultured in Whitten's medium. Mature follicles were present at the earliest time examined (6 h). Thirty-eight percent of females that had been paired for < 12 h after the first copulation ovulated, whereas all females paired >/= 12 h after the first copulation ovulated. Virtually all (> 99%) oocytes recovered from females paired for >/= 12 h after first copulation were fertilized. Pairing time after first copulation and mean copulation-bout duration were significant (p < 0.05) determinants of embryo number. Embryos entered the uterine horns and implanted on Days 3 and 4, respectively, after first copulation (Day 0). Embryos cultured in vitro underwent approximately one cell division per day, a rate similar to that in vivo. We conclude that prairie voles ovulate reliably after pairing for >/= 12 h, although some females showed exceptional sensitivity not predicted by the variables quantified. Prolonged mating for longer than 12 h increased the total embryos produced. This mechanism likely has adaptive significance for increasing offspring number.     

Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium

At least 71% of CF1 x B6SJLF1/J embryos developed from the 1-cell stage to the blastocyst stage in an optimum glutamine concentration of 1 mM, as long as glucose was present after the first 48 h of culture. Blastocysts raised under these conditions had significantly more cells than did blastocysts raised in CZB medium alone (glutamine present, glucose absent). Embryos raised in vivo accumulated 170-200 fmol glutamine/embryo/h at the unfertilized egg and 1-cell stages with a decline to 145 fmol/embryo/h at the 2-cell stage, followed by sharp increases to 400 and 850 fmol/embryo/h at the 8-cell and blastocyst stages. The presence or absence of glucose in the labelling medium had no effect on glutamine uptake by these embryos. Embryos raised in vitro accumulated 2-3 times more glutamine at stages comparable to those of embryos raised in vivo. In all cases in which 1-cell to blastocyst development in vitro was successful, glucose was present in the culture medium and the incremental uptake of glutamine between the 8-cell stage and the blastocyst stage was approximately 2-fold. This was also the increment for in-vivo raised embryos. When glucose was not present after the first 48 h, the 8-cell to blastocyst glutamine increment was not significant, and development into blastocysts was reduced. The results also show that glutamine can be used as an energy source for the generation of CO2 through the TCA cycle by all stages of preimplantation mouse development, whether raised in vivo or in vitro from the 1-cell stage. Two-cell embryos raised in vivo converted as much as 70% of the glutamine uptake into CO2, consistent with an important role for glutamine in the very earliest stages of preimplantation development. Cultured blastocysts appeared to convert less glutamine and the presence of glucose in the culture medium seemed to inhibit this conversion.     

Glucose, glutamine and inorganic phosphate in early development of the pig embryo in vitro

Pig embryos at the 1- or 2-cell stage (before the 'block' to development in vitro) were cultured in 8 different media derived from Krebs'-Ringer-bicarbonate medium. A 2 x 2 x 2 factorial arrangement was used for the treatments, with glucose, glutamine and phosphate being the major effects tested. Embryos were obtained from sows approximately 44-48 h after the observation of oestrus, with the majority being at the 1-cell stage. Embryos from each female were randomly assigned to each treatment. After in-vitro culture, all embryos were scored for the stage of development attained and stained to determine final cell number. Significant effects were evident due to female, glucose, glutamine, a phosphate x glucose interaction and a glutamine x glucose interaction. None of the media components tested was inhibitory to embryo development. The greatest development (45-60% morula or blastocyst) was achieved with glucose and glutamine (both alone and in combination) in the media, demonstrating that an amino acid can serve as the sole energy source for complete preimplantation embryonic development in vitro.     

Ovulation and Embryonic Developmental Rate Following hCG-stimulation in Sows

The hCG induced ovulation in sows was studied by use of ultrasonography, and an investigation of the development and diversity of the zygotes/embryos was performed at 24 h after ovulation. Crossbred sows (N=48) were weaned (day 0) and checked for heat twice daily from day 3 onwards. From day 4, the ovaries were transrectally scanned twice daily On day 4, the sows were given an injection of 750 iu hCG im and inseminated 27 ± 2 h (X ± SD) and 38 ± 1 h later. From 38 to 48 h after the hCG injection, the ovaries were scanned at 60 to 90 min intervals. At 24 h after ovulation the oviducts were surgically flushed in 18 sows. Out of the 48 sows, 34 showed heat at 12–36 h after the hCG-treatment and 14 showed heat before the hCG treatment. In the former group of sows, 20 (59%) ovulated within the interval of 38 to 48 h after the hCG treatment, and the follicular size immediately before ovulation was 7.8 ± 0.6 mm. Among the sows which showed heat before hCG treatment only 7 (50%) ovulated within the above interval and the preovulatory follicle size was larger (8.3 ± 0.5, p<0.05) than in the former group of sows, which showed heat after the hCG treatment. The flushing of 18 sows yielded a total of 243 ova, 70 (29 %) 1-cell stages, 160 (66 %) 2-cell stages and 13 (5%) 4-cell stages. A pronounced difference in the degree of variation in embryonic development was seen between sows: 4 animals yielded 1- to 4-cell stages, one exclusively 2-cell stage. In conclusion, the control of ovulation in sows by hCG treatment will affect the follicular growth and the exact timing of ovulation can not always be relied on. It is strongly recommended to use ultrasonography to monitor the time of ovulation if this parameter is important. Ova recovered at 24±1 h after the median time of ovulation revealed a pronounced diversity (1- to 4- cell stage) within sows. No obvious relation with this embryonic diversity and the follicular size at ovulation was seen in these data.     

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.     

Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but ininjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.     

Early embryonic development and in vitro culture of in vivo produced embryos in the farmed European polecat (Mustela putorius)

Early embryonic development and in vitro culture of in vivo produced embryos in the farmed European polecat (Mustela putorius) was investigated as a part of an ex situ conservation program of the endangered European mink (Mustela lutreola), using the European polecat as a model species. The oestrus cycles of 34 yearling polecat females were monitored by visual examination of the vulval swelling and, to induce ovulation, the females were mated once daily on two consecutive days. Sixteen yearling males were used for mating. The females were humanely killed 3-14 days after the first mating and the uteri and oviducts were collected for embryo recovery. Uterine and oviductal flushings yielded a total number of 295 embryos, representing developmental stages from the 1-cell stage to large expanded and hatched blastocysts. On Day 3 after the first mating, only 1-16-cell stage embryos were recovered. Between Days 4 and 6 after the first mating, 1-16-cell stage embryos and morulae were found. The first blastocysts were recovered on Day 7 after the first mating. The first implanted blastocysts were detected on Day 11 after the first mating. A total number of 85 embryos were in vitro cultured after recovery. Blastocyst production rates for in vitro cultured 1-16-cell stage embryos and for morulae/compact morulae were 68 and 84%, respectively. For all cultured embryos, the hatching rate was 15%. The in vitro culture requirements for the preimplantation embryos of the farmed European polecat remain to be determined before further utilization of the technique.     

Effect of gossypol on bovine embryo development during the preimplantation period

The purpose of this study was to evaluate the effect of varying doses of gossypol acetic acid on early bovine embryo development in vitro. One hundred and forty-eight excellent and good quality bovine morulae were randomly cultured in 0, 1.0, 5.0, 10.0, 30.0 mug gossypol acetic acid (GAA) in normal steer serum and Ham's F-10 media. Bovine embryo development was assessed at 12-h intervals for 96 h. Sixty-seven percent of embryos developed in 0 mug GAA to the hatched blastocyst stage, while 43, 19, 4 and 0% had comparable development in 1.0, 5.0, 10.0 and 30.0 mug GAA, respectively. Embryos in 5.0 mug GAA had a delayed development to the blastocyst stage compared to embryos in 1.0 mug GAA. Development time to expanded blastocyst stage was longer for 10.0 mug GAA embryos than 0, and 1.0 GAA-treated embryos. No embryo cultured in 30.0 mug GAA advanced past the morula stage. Final developmental scores were highest for embryos in 0 mug GAA (4.06) and lowest for embryos cultured in 10.0 and 30.0 mug GAA (0.44 and -0.02, respectively). Embryos cultured in higher doses of GAA degenerated sooner than embryos cultured in 0 mug GAA. These data show a dose-dependent detrimental action of GAA on early bovine embryo development and suggest a direct action on the embryo itself.     

Effect of repeated ACTH-stimulation on early embryonic development and hormonal profiles in sows

This study was conducted to evaluate the effect of adrenal stimulation by synthetic adrenocorticotrophic hormone (ACTH) on the first 2 days of pregnancy in 22 multiparous sows. The experiment was performed during the second oestrus after weaning and the sows were divided into one control (C-group) and one experiment group (E-group). To determine the time of ovulation, transrectal ultrasonographic examination was performed. E-group sows were treated repeatedly with 0.1 mg/kg bodyweight of synthetic ACTH (tetracosactide) i.v. 4-8h after ovulation and continuing every 6h, until slaughter. Blood samples were collected every second hour from about 12h before expected ovulation until slaughter and were analyzed for cortisol, prostaglandin F(2 alpha) -metabolite, and progesterone (P(4)). All sows were slaughtered approximately 48 h after ovulation and the isthmic part of the oviduct was divided into three equally long segments and flushed separately with phosphate buffered saline (PBS). The uterine horns were also flushed with PBS. The embryos of the E-group sows tended (P=0.056) to have a lower cleavage rate than the embryos of the C-group sows but there was no difference between groups in oviductal transport rate of the embryos. In the E-group, significantly (P<0.05) more sows had only embryos with <20 spermatozoa attached to the ZP compared with the C-group. The plasma concentration of cortisol was significantly higher (P<0.0001) in the E-group sows during the time of treatment while the baseline level of prostaglandin F(2 alpha) -metabolite was significantly lower. The baseline level of progesterone increased in both groups after ovulation but there was no significant difference between the groups. Repeated ACTH-stimulation (1) had no effect on the oviductal transport rate of the embryos, (2) had a negative effect on the embryo development, (3) and caused a changed endocrine profile that might have changed oviductal milieu affecting embryo development.     

Cryopreservation of embryos as a means of germ plasm conservation in sheep

Embryos were collected from 4 lines of Targhee sheep between 1986 and 1990. The lines were selected for preweaning growth rate (Lines DH and HW) or for multiple births (Line HT); Line C served as an unselected control group. Estrus was synchronized using fluorogestone acetate-impregnated vaginal pessaries, and ewes were superovulated with FSH. Embryos at the morula or blastocyst stage were surgically recovered from mature ewes at Days 5 to 6 and were frozen following morphological evaluation. The overall average number of freezable embryos per collection was 2.9, and did not differ significantly among years or among lines. Of the embryos collected between 1986 and 1988, 92 were transferred to 53 recipients in 1989, producing 53 lambs. Survival rates were 60.9 and 47.8%, respectively, for embryos evaluated as good and fair after thawing. Good-quality blastocysts yielded the highest survival rate (64.4%). Analyses indicated no significant effects of line, developmental stage or embryo evaluation on the incidence of lambing. It was concluded that embryos of morula or blastocyst stage can be successfully frozen for extended periods. The data on embryo yield and survival following cryopreservation were used to calculate numbers of donors needed to preserve and reconstitute a population of specified size.     

Bovine embryo morphology and evaluation

The following paper briefly reviews the morphology of the bovine embryo and presents a retrospective analysis of bovine embryo transfer results accumulated from April to December of 1982 at a commercial embryo transfer center. Of particular interests were bovine embryo morphology, assessment of embryo quality, and recipient-donor, recipient-embryo synchrony requirements. Embryos were recovered from superovulated donors five to nine days after estrus (estrus = day O). All embryos were individually examined at 200X for cell stage of development and embryo quality. Embryos were nonsurgically transferred to recipients that were within two days of estrous cycle synchrony with the donor. Attempts were made to synchronize estimated developmental age of embryos to the day of the recipient cycle. A high degree of variability was observed in morphological development and embryo quality within and among donors. Embryo recovery in individual donors resulted in a wide range of embryonic cell stages, often differing in estimated developmental ages from 24 to 48 hours. A total of 783 embryos were transferred, resulting in 308 pregnancies. Stage of embryonic development (16-cell through hatched blastocyst) had little effect on pregnancy rates. Embryo quality was a more accurate predictor of success. Embryos of excellent, good, fair and poor categories resulted in 45%, 44%, 27% and 20% pregnancy rates, respectively. Recipient-donor estrous cycle synchrony of two days in either direction did not significantly alter pregnancy rates. However, 88% of 258 pregnancies (584 total transfers) occurred with a +/-1 day recipient-embryo synchrony compared to 74% based on +/-1 day recipient-donor cycle synchrony (P<0.001). Results suggest that transfer of bovine embryos based on synchrony between day of recipient cycle and state of embryonic development provides higher pregnancy rates than transfers based on recipient-donor cycle synchrony.     

Differences between Brahman and Holstein cows in response to estrus synchronization,superovulation and resistance of embryos to heat shock

Embryos from Bos indicus are more resistant to elevated culture temperature (i.e. heat shock) than embryos from some Bos taurus breeds. The present experiment was designed to determine if Brahman embryos have greater resistance to heat shock than Holstein embryos at a stage in development before the embryonic genome was fully activated. A second objective was to test breed effects on estrus synchronization and superovulation responses. A total of 29 Brahman and 24 Holstein cows were subjected to estrus synchronization using gonadotropin releasing hormone (GnRH) and prostaglandin F2alpha (PGF2alpha) superovulation. Embryos were collected at 48 h and day 5 after insemination. There was a tendency for a lower proportion of Brahmans to be detected in standing estrus than Holsteins. There were no differences between breeds in the proportion of cows detected in estrus using both tailpaint and standing estrus as criteria or in interval from PGF2alpha to estrus. The degree of synchrony in estrus was greater for Brahmans. Superovulation response was generally similar between breeds. At 48 h after insemination, there was a tendency for a greater proportion of Brahman oocytes to have undergone cleavage. Uncleaved oocytes were cultured for an additional 24 h-at this time, cleavage rate was similar between breeds. When embryos reached the 2-4-cell stage, they were heat-shocked for 4.5 h at 41 degrees C. This heat shock reduced the proportion of embryos that developed to the blastocyst stage but there was no breedxtreatment interaction. At day 5 after insemination, the number of embryos recovered was too low to allow comparison of breed effects. In conclusion, genetic effects on cellular thermotolerance that make Brahman embryos more resistant to heat shock are not expressed at the 2-4-cell stage. There were few differences between Brahman and Holstein in response to estrus synchronization and superovulation. The fact that cleavage tended to occur earlier in Brahman than Holstein embryos suggests breed differences in timing of ovulation, fertilization or events leading to cleavage.     

Recovery rate and quality of embryos from mares inseminated after ovulation

The aim of this study was to evaluate the quality of embryos and their recovery rate from mares inseminated at different intervals after ovulation. Finnhorse and warmblood mares were inseminated with fresh semen 8 to 16 h, 16 to 24 h, or 24 to 32 h after ovulation. Control mares were inseminated before ovulation. Sixty-seven embryo flushings were performed between Days 7 and 9 after ovulation/insemination. Thirteen mares were not flushed, but their uteri were scanned for pregnancy on Days 14 to 16. Embryo recovery rates decreased as time from ovulation to insemination increased, although embryo quality remained normal as evaluated by morphological criteria and mitotic index. However, postovulatory insemination in this trial appeared to delay embryo development, since the embryos recovered from mares inseminated after ovulation were appreciably smaller and at an earlier stage of development than control embryos recovered from mares inseminated prior to ovulation. Part of this delay in embryo development in the postovulation group could be due to the time needed for sperm capacitation. In addition, as the time from ovulation to insemination increased, embryo development might have been further delayed by defects in the aging oocyte.     

High overall in vitro efficiency of porcine handmade cloning (HMC) combining partial zona digestion and oocyte trisection with sequential culture

We investigated the in vitro developmental competence of porcine embryos produced from in vitro matured (IVM) oocytes by improved HMC and parthenogenetic activation (PA). Embryos were cultured in a modified North Carolina State University (NCSU37) medium. Firstly, we compared the developmental competence between oocytes from sows and gilts by zona-intact (ZI) and zona-free (ZF) PA. Significantly higher (p < 0.05) blastocyst rates were obtained from sow oocytes (42 +/- 4% for ZF and 55 +/- 6% for ZI) than gilt oocytes (20 +/- 2% for ZF and 26 +/- 5% for ZI). Secondly, sow oocytes were used to establish the modified HMC that was based on a modified enucleation with partial zona digestion and trisection of porcine oocytes and the use of three cytoplasts and one somatic cell for embryo reconstruction. In vitro fertilization (IVF) and in parallel ZF PA were used as the control systems. After oocyte trisection, >90% of oocyte fragments were recovered, resulting in an average of 37 reconstructed embryos from 100 oocytes. Blastocyst rates of HMC, IVF, and ZF PA embryos were 17 +/- 4%, 30 +/- 6%, and 47 +/- 4%, respectively. Our results prove that HMC in pigs may result in high in vitro efficiency up until the blastocyst stage. In vivo developmental competence will be confirmed in embryo transfer experiments.     

In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.