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There are few reports describing the use of Doppler for reproductive imaging in large animals; only one study (in mares) assessed umbilical blood flow (Bollwein et al. 2004 Theriogenology 61, 499–509). Conditions that constrict the placental vascular bed (i.e. hemorrhage, thrombosis, abnormal development, etc.) increase resistance to incoming blood. Thus, blood flow in umbilical arteries can be used to monitor placental development and function (Giles et al. 1985 Brit. J. Obstet. Gynaecol. 92, 31–38). The objective of the present study was to characterize the Doppler flow velocity waveform in umbilical arteries of cows with apparently normal pregnancies. Twenty-three multiparous, nonlactating Aberdeen Angus cows with pregnancies achieved after transfer of embryos derived by IVF (n = 10) or multiple ovulation and embryo transfer (MOET) (n = 13) were examined weekly from 5 to 38 weeks of gestation. Ultrasonography (Toshiba Nemio 20, Tokyo, Japan) was done using a 5–10 MHz intraoperative finger transducer (transrectal) from 5 to 17 weeks of pregnancy and thereafter using a 3–6 MHz linear-array transducer (transabdominal). The interrogation angle (between the ultrasound beam and the artery) ranged from 45° to 60°. Three resistance indices were calculated: A/B ratio, Resistance Index (RI) = (A − B)/A, and Pulsatility Index (PI) = (A − B)/M. [A = systole, B = diastole, and M = mean maximum Doppler-Shift frequency over the cardiac cycle.] The mean ± SEM duration of pregnancy was 285 ± 1.6 days (range: 269 to 291 days). A repeated measure ANOVA was used to detect differences between groups for every week using the Wald-Wolfowitz test (InfoStat V1.5, FCA, Universidad de Córdoba, Córdoba, Argentina). All 3 resistance indices decreased (by >50%) until 26 weeks, with no substantial change thereafter (Table 1). From 5 to 18 weeks of pregnancy, blood flow was characterized by a systolic pattern (i.e. high resistance with absence of diastolic flow); at 20 weeks, all fetuses had a diastolic flow, consistent with low resistance. There were differences (P < 0.05) between IVF- and MOET-derived pregnancies for RI and PI at 38 and 26 weeks of gestation, respectively, indicating differences in the placental vascular development at these weeks of gestation. In conclusion, umbilical cord blood flow in cattle was characterized by high resistance (5 weeks of gestation), but resistance decreased until 26 weeks; consequently, blood flow was initially systolic but became diastolic. Doppler sonography was useful for assessment of umbilical blood flow from 5 to 38 weeks of pregnancy, and may be useful for assessing placental function in pregnancies under risk, e.g. clone-derived pregnancies (Bertolini et al. 2002 Theriogenology 57, 181–187). Table 1. RI and PI at 19, 22, 26, 30, and 38 weeks of pregnancy in IVF- and MOET-derived pregnancies (mean ± sd)

Sperm sexing has become a world-wide technology, now available in many countries. The method has been incorporated into many reproductive technologies such as embryo production (Zhang et al. 2003 Theriogenology 60, 1657–1663), but sex-sorting is limited when bulls are located far from sorters or when only frozen semen is available. Previous studies on sexing frozen–thawed spermatozoa have been done in rams, which resulted in retention of the spermatozoan functional capacities (Hollinshead et al. 2004 Reproduction 127, 557–568). In vitro characteristics were assessed in bulls after sexing of thawed sperm (Hollinshead et al. 2004 Theriogenology 62, 958–968); however, the fertilizing capacity of frozen–thawed sex-sorted (FTSS) spermatozoa was not tested. The aim of the present study was to compare cleavage and embryo development rate among frozen–thawed (FT), sex-sorted frozen–thawed (SSFT), and FTSS bull spermatozoa. For FT, sperm were diluted to a final concentration of 60 × 106 sperm/mL, packaged in 0.5-mL straws, and frozen. In SSFT, spermatozoa were sex-sorted by flow cytometry following Schenk protocols (1999 Theriogenology 52, 1375–1391). Three × 106 spermatozoa were packaged into 0.25-mL straws and frozen. The final treatment (FTSS) consisted of thawing 6 to 10 frozen straws of 4 different bulls containing an average of 25 × 106 spermatozoa and centrifuging at 600g for 15 min at 21°C to extract cryodiluent. Spermatozoa were diluted and stained with Hoechst 33342 (stain concentration of 112.5 µM, the same used for SSFT treatment) following Schenk sexed-semen protocols (1999), sex-sorted by a flow cytometer, and collected in Tris-base extender containing 20% egg yolk. For each ejaculate frozen–thawed, SSFT and FTSS spermatozoa were prepared for oocyte in vitro fertilization. Also, semen from a bull routinely used as a control in the laboratory was added for a better comparison of results. Oocytes from a slaughterhouse were processed following standard in vitro fertilization procedures (Ferré 2002 Theriogenology 57, 664) 4 times for each bull, and comparison was made between treatments. Results were analyzed by ANOVA. No significant differences were observed among bulls (data not shown) (P > 0.05). Although embryo development rate was statistically different between sexed and non-sexed groups (P < 0.05), results showed that frozen–thawed bull spermatozoa can be sex-sorted and used for in vitro fertilization with comparable developmental rates comparable to those when frozen sexed semen is used (Table 1). This opens a new commercial window for cases where pre-selected sexed embryos from bulls that are not in AI centers are desired, also giving an opportunity for dead bulls. Nevertheless, since a large number of straws are necessary, further studies must be carried out to make this procedure more efficient and economically profitable. Table 1. Results of cleavage and embryo development rates between spermatozoa treatments

In ruminants, abnormal increased liver size is a common description cited in many postmortem studies performed on aborted fetuses and stillborn cloned calves (Heyman et al. 2002 Biol. Reprod. 66, 6–13). Although fetal liver size can be accurately determined throughout pregnancy using ultrasonography as a method of monitoring fetal health in humans, there are no reports of this being done in cattle. Thus, the aim of this study was to prospectively characterize the ultrasonographic fetal liver growth pattern in IVF-derived pregnancies, and then to establish comparisons with measurements taken from cloned fetuses. For this purpose, (A) IVF-derived pregnancies were used as the control group (n = 10), and the cloned ones were split into 2 groups according to the outcome of their pregnancies: (B) clones that died between 110 and 282 days (n = 21), and (C) clones born alive (n = 16). All recipients were multiparous, nonlactating, and Aberdeen Angus breed. Measurements were done by ultrasonography (Toshiba Nemio 20, Tokyo, Japan) using a 5–10 MHz intraoperative finger probe from 72 to 114 days (transrectal) and a 3–6 MHz linear-array probe between 143 and 212 days (transabdominal). Three parameters of liver size were measured: (a) in a coronal (longitudinal) plane of the fetus, cephalocaudal diameter (CC) was taken from the dome of the right hemi-diaphragm to the tip of the right lobe; (b) in an axial (transverse) plane at right angles to one another passing through or slightly caudal to the portal umbilical venous complex: (b1) transverse diameter (TD) and (b2) sagittal diameter (SD) were determined (Gimondo et al. 1995 J. Ultrasound Med. 14, 327–333). A repeated-measure ANOVA detected significant interaction for the 3 variables included in this study (P < 0.01); therefore, differences between groups at each week of gestation were analyzed using the Wald-Wolfowitz test (InfoStat V1.5; FCA, Universidad de Córdoba, Córdoba, Argentina). Results showed that CC, TD, and SD were statistically smaller in the IVF group throughout pregnancy (P < 0.05). No significant differences between (B) and (C) groups were found at 72 and 87 days (P > 0.05). However, from 143 days onward, liver of fetuses from the (B) group were statistically larger in CC, TD, and SD diameters than those values obtained in the (A) and (C) groups (P < 0.05) (Table 1). To conclude, the present study showed that fetal liver size can be measured throughout gestation in cows using noninvasive ultrasonography. Secondly, since fetuses from (A) and (C) groups were born alive, increased abnormal size measurements after 140 days can be used as indicator of clones that fail late in gestation (B). Also, this methodology may enhance sonographic assessment of fetal growth abnormalities and conditions with fetal liver involvement. Further studies will be necessary to evaluate differences between naturally conceived and IVF fetuses and the correspondence of this three-dimensional evaluation of the liver and to estimate its weight. Table 1. TD and SD at 72, 143, and 212 days of gestation in (A), (B), and (C) groups (n = 16) (mean ± SD)

In embryos reconstructed by somatic cell nuclear transfer (SCNT), components of the oocyte cytoplasm are capable of reprogramming the somatic genome to control subsequent development. Although the mechanisms that control nuclear reprogramming are unknown, we have previously hypothesized that the occurrence of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the donor nucleus are beneficial. In previous studies we have demonstrated that treatment of ovine oocytes with caffeine (10 mM), a protein phosphatase inhibitor, increased the activities of both MPF and MAPK in enucleated oocytes (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) and additionally resulted in a significant increase in the occurrence of NEBD and PCC in donor nuclei. Furthermore, SCNT embryos reconstructed following caffeine treatment had significantly increased cell numbers at the blastocyst stage. More recently we have demonstrated that the use of caffeine treated ovine oocytes as cytoplast recipients can regulate the expression of several developmentally important genes in SCNT embryos, including Oct-4 and interferon-tau (Choi et al. 2006 Reprod. Fertil. Dev. 18, in press). This study was designed to establish the developmental potential of NT embryos reconstructed using caffeine treated oocytes as cytoplast recipients. Ear skin fibroblast cells established from a Merino ram were quiesced in DMEM containing 0.1% fetal bovine serum (FBS) for 3 days. Oocyte maturation and embryo reconstruction and culture were performed as previously described (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) with the exception that ovaries from Merino � Romney Marsh cross ewes were stimulated with FSH sponge (Folltropin�-V; Bioniche Animal Health, Beltsville, Ontario, Canada) and were collected at slaughter on Day 13 following sponging. Blastocyst stage embryos were surgically transferred to the uterine horn of synchronized Merino � Romney Marsh cross recipients (three blastocysts per recipient). Recipient ewes were scanned by ultrasonography at Days 30, 60, and 90 following embryo transfer. All data were analyzed by chi-square test. There were no differences in fusion (145/167; 86.8% vs. 174/205; 84.9%), cleavage (123/145; 84.8% vs. 135/174; 77.6%), or the development to blastocyst (33/145; 22.8% vs. 34/174; 19.5%) between control SCNT embryos and caffeine treated SCNT embryos. However, although the frequency of pregnancy between control and caffeine-treated NT groups (5/15; 33.3% vs. 7/14; 50.0%) at 30 days was not significantly different, control SCNT embryos showed significantly lower pregnancies (1/15; 6.7%) than caffeine treated SCNT embryos (4/14; 28.6%) at both 60 and 90 days. In conclusion, embryos reconstructed using caffeine-treated cytoplasts can induce pregnancy at the same frequency as untreated controls; furthermore, the results suggest that SCNT embryos produced in this way are more able to maintain pregnancy.

In ruminants, the embryonic vesicle is composed of the yolk and the allantoic and amniotic sacs. In cattle, the allantoic sac represents the largest fluid accumulation in the uterus from early pregnancy onward, but is irregular in shape as it conforms to the uterine horns. It is, therefore, difficult to ultrasonographically measure the allantoic compartment. Conversely, the amniotic sac remains ellipsoidal in shape and can be evaluated more easily. The aim of this study was to characterize the development of the amniotic sac in relation to embryo development. Thirteen multiparous, nonlactating Aberdeen Angus cows were examined weekly from 30 days to 72 days of gestation. Epidural anesthesia was induced (6 mL of 2% lidocaine) to obtain light relaxation of the rectum and prevent straining. Measurements were taken by ultrasonography (Toshiba Nemio 20, Tokyo, Japan) using a 5–10 MHz intraoperative finger probe from 30 days to 58 days of pregnancy, and a 3–6 MHz linear-array probe thereafter. Length (D1) was registered in a longitudinal section of the amniotic sac, whereas width (D2) and height (D3) were measured in a cross section. Volume (cm3) was calculated with the formula 4 ÷ 3 × Pi × (D1 ÷ 2) × (D2 ÷ 2) × (D3 ÷ 2) ÷ 1000 and mean amniotic diameter (MAD) was calculated as the average of the three measurements (Hellman et al. 1969 Obstet. Gynecol. 103, 789). Crown rump length (CRL) was measured in a longitudinal section of the embryo when it was in a neutral position. Results show that volume, MAD, and the CRL increase progressively during the first trimester (Table 1). In the first 50 days of gestation, the difference (MAD-CRL) changed from a negative to a positive value, probably as a reflection of increasing embryonic production of amniotic fluid (Wallenburg et al. 1988 Med. 5, 191). From 58 days of gestation onward, the difference became progressively more negative due to a faster relative growth rate of the embryo than of the amniotic sac. To conclude, the present table may be useful in distinguishing between normal and abnormal early pregnancy, including embryonic growth retardation and hydrops amnios (Wintour 1986 et al. Aust. Vet. J. 216, 221). Table 1. Mean amniotic diameter (MAD), crown rump length (CRL), and the difference (MAD-CRL) during pregnancy in cattle (mean ± SD; n = 13)

The most important factor in bovine embryo transfer programs is the low efficiency in the utilization of the recipients; this low efficiency is associated with low response to synchronization protocols and failures in estrus detection. It has been shown that cows transferred at fixed time with in vivo-derived embryos resulted in high rates of recipients selected for transfer and high overall pregnancy rates (recipients pregnant/recipients treated) (Tribulo et al. 2002 Theriogenology 57, 563). An experiment was designed to evaluate the pregnancy rate in recipients transferred with in vivo (fresh and frozen), IVF, and cloned-derived embryos without estrus detection. A total of 1555 non-lactating Bos Taurus crossbred beef cows was divided into two groups. Cows from group 1 (n = 421) were synchronized with a progesterone intravaginal releasing device (1 g P4; DIB, Syntex®, Buenos Aires, Argentina) plus 2 mg of estradiol benzoate (EB) i.m. (Syntex®) on Day 0. On Day 5, they received 400 IU of eCG (Novormon 5000, Syntex®) i.m. and 150 μg of D-Cloprostenol (PGF2α) (Bioprost-D, Biotay®, Buenos Aires, Argentina). The DIB devices were removed on Day 8 and on Day 9, 1 mg of EB was injected. Day 10 was arbitrarily considered as the day of estrus. Cows from group 2 (n = 1134) received 2 doses of PGF2α 14 days apart and were checked for heat during 5 days after the second PGF2α dose. Cows of both groups were examined 7 days after estrus by ultrasonography (Pie Medical Scanner 200®) and those with a corpus luteum >10 mm of diameter were transferred nonsurgically with in vivo (fresh and frozen), IVF, and cloned-derived embryos. In group 1, 360 cows were transferred, and in group 2, 726 cows were transferred (Table 1). Pregnancy was diagnosed 23 days later by ultrasonography (Pie Medical Scanner 200®). The pregnancy rates were compared statistically between groups 1 and 2 by analysis of variance (Infostat, LSD Fisher). There was no significant statistic difference (P > 0.05) between pregnancy rate in group 1 and 2 with in vivo (fresh), IVF, and cloned-derived embryos. However, pregnancy rate of frozen in vivo-derived embryos was lower in group 1 than in group 2 (P < 0.05). Results showed that treatment using DIB combined with EB, PGF2α, and eCG associated with embryo transfer without estrus detection (group 1) had no difference in pregnancy rate when compared with the treatment where synchronization with PGF2α and heat detection were used (group 2). Another important advantage is the use the group 1 treatment for increasing the flexibility and efficiency in the management of the recipients of in vivo, IVF, and cloned-derived embryo transfer programs. Table 1. Comparison of pregnancy rates between group 1 (embryo transfer at fixed time) and group 2 (embryo transfer 7 days after estrus detection)

Cell line cryopreservation is nowadays one of the most useful tools in somatic cell nuclear transfer. Although this technique guarantees the genetic storage for an unlimited period of time, many studies have shown that it produces different kinds of cellular damage such as DNA fragmentation (Men et al. 2003 Mol. Reprod. Dev. 64, 245–250) and ultrastructural cell anomalies (Taddei et al. 2001 Cryobiology 42, 244–555; Nardid et al. 1997 Cryobiology 34, 107–113). The aim of the present study was to evaluate how repeated cell freezing/thawing processes could affect the pregnancy rate of bovine nuclear transfer-derived embryos. Two adult fibroblast cell lines from different animals were separated into two groups according to the number of freezing/thawing processes they went through (1 vs. 3). For both groups, the first freezing process was performed with cells from passage 1. Cells from passages 3 and 4 were used for the second and third freezings, respectively. The time interval between thawing and next freezing was 20 days. Cells were harvested at 80% confluence using trypsin, and cryopreservation was performed in D-MEM with 35% FCS and 10% DMSO. Enucleation and nuclear transfer (NT) were performed as described by Cibelli et al. (1998 Science 280, 1256–1258) with modifications. For both groups, cells from the same number of passages were used for the NT assays (between passages 2 and 7). Cytoplasts were activated using 5 μM ionomycin for 4 min and the couplets were subsequently fused. The fused units were cultured in 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B for 6 h. Embryo culture was performed at 38.5°C in a 5% O2, 5% CO2, 90% N2 atmosphere, in 50-μL drops of KSOM. On Day 3 of culture, the KSOM was supplemented with 2% FCS and 0.2 mM glucose. After 6–7 days, the embryos were non surgically transferred to synchronized recipients. Pregnancy at 30 and 60 days was recorded by ultrasonography using an Aloka 500® scanner (Aloka Co., Tokyo, Japan). Data were analyzed by ANOVA (InfoStat, Austin, TX, USA) (Table 1). The results show an association between the number of cell freezing/thawing processes and a higher pregnancy loss at 60 days. This could be related to the cellular damages caused by multiple cryopreservation procedures, which could lead to chromosomal abnormalities in the donor cells and thus in the nuclear transfer (NT) embryos and pregnancies derived from them. Further studies should be done in order to evaluate the chromosomal status of the cell lines used in this work. Table 1. Numbers of cell freezings/thawings and their effect on pregnancy rate of bovine NT-derived embryos

According to the production objectives of beef or dairy commercial farms, the possibility of gender selection of the offspring is an important tool to optimize the use of genetic and economical resources. The use of sexed sorted semen by flow cytometry to inseminate donors in a MOET program has successfully demonstrated a normal embryo production per cow (Theriogenology 59:513). The aim of this study was to evaluate the pregnancy rate and sex accuracy obtained after transferring embryos collected using sexed semen to inseminate the donors in MOET programs. The semen was provided by our commercial laboratory. Eighteen cows were inseminated with frozen female sexed semen and 22 with frozen male sexed semen. Embryos were collected using a commercial flushing medium with BSA and antibiotics. After the embryos were washed through 10 drops of holding medium, 50 embryos were transferred fresh;; 150 were frozen in 1.5M ethylene glycol, thawed 10 seconds in air;; and held for 30 seconds in a water bath at 35°C before transferring them. Fresh embryo stage and quality rates were: morula grade 1: 49%, grade 2: 13%; blastocyst grade 1: 34%, grade 2: 4%. Frozen embryo stage and quality rates were: morulae grade 1: 28%, grade 2: 18%; blastocyst grade 1: 47%, grade 2: 7%. The embryo transfers (ET) were performed in previously synchronized recipients injected with two doses of prostaglandin 12 days apart. Thirty days after ET, the diagnosis of pregnancy was done by transrectal ultrasonography (ALOKA 500 Scanner) to asses the pregnancy rate. Sixty days after the ET, fetal sex determination was done. The results are shown in the table 1 below. The pregnancy rates in both fresh and frozen-thawed preselected sex embryos are similar to those obtained with nonpreselected sex embryos (Theriogenology 56:1401). The high rate of sex accuracy shows the reliability of the technique of sexed sorted semen by flow cytometry to produce preselected sex embryos in superovulated cows. This research was supported by Fundación Margarita Perez Companc. Table 1