D.K. Berg

Trophectoderm Lineage Determination in Cattle

The trophectoderm (TE) and inner cell mass (ICM) are committed and marked by reciprocal expression of Cdx2 and Oct4 in mouse late blastocysts. We find that the TE is not committed at equivalent stages in cattle, and that bovine Cdx2 is required later, for TE maintenance, but does not repress Oct4 expression. A mouse Oct4 (mOct4) reporter, repressed in mouse TE, remained active in the cattle TE; bovine Oct4 constructs were not repressed in the mouse TE. mOct4 has acquired Tcfap2 binding sites mediating Cdx2-independent repression-cattle, humans, and rabbits do not contain these sites and maintain high Oct4 levels in the TE. Our data suggest that the regulatory circuitry determining ICM/TE identity has been rewired in mice, to allow rapid TE differentiation and early blastocyst implantation. These findings thus emphasize ways in which mice may not be representative of the earliest stages of mammalian development and stem cell biology.

Storage of semen and artificial insemination in deer

Methods of collection and freezing of semen of some deer species and aspects of controlled reproduction associated with the use of frozen-thawed semen by artificial insemination (AI) are discussed.

Embryo loss in cattle between Days 7 and 16 of pregnancy

Embryo loss between embryonic Days 7 and 16 (Day 0=day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P=0.03) as more embryos were transferred, particularly at later stages (Day 14, P<0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P<0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P<0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P<0.05; winter-spring, P<0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).

Red Deer Cloned from Antler Stem Cells and Their Differentiated Progeny

Abstract The significance of donor cell differentiation status for successful cloning by somatic cell nuclear transfer (SCNT) is unclear. Here, we cloned a new species, red deer (Cervus elaphus), from multipotent antler stem cells and their differentiated progeny. Cultured donor cell lines from male antlerogenic periosteum (AP) were left undifferentiated or chemically induced to initiate osteogenesis or adipogenesis. Based on their morphology and marker gene expression profile, donor cells were classified as undifferentiated AP cells, presumptive osteoblasts, or adipocytes. Adipocytes upregulated adipogenic markers procollagen type I alpha 2 (COL1A2), peroxisome proliferator-activated receptor gamma 2 (PPARG), and gylceraldehyde-3-phosphate dehydrogenase (GAPDH), and downregulated antlerogenic transcripts POU-domain class 5 transcription factor (POU5F1) and parathyroid hormone (PTH)-like hormone (PTHLH). Despite differences prior to NT, transcript abundance of donor-specific markers COL1A2, PPARG, GAPDH, and POU5F1 did not differ significantly in cloned blastocysts (P = 0.10, 0.50, 0.61, and 0.16, respectively). However, donor cell and blastocyst expression levels were completely different for most genes analyzed, indicating their successful reprogramming. The type of donor cell used for NT (AP, bone, and fat cells), had no effect on in vitro development to blastocysts (93 [38%] of 248 vs. 32 [44%] of 73 vs. 59 [32%] of 183, respectively). Likewise, development to weaning was not significantly different between the three cell types (2 [4%] of 46 vs. 2 [29%] of 7 vs. 4 [13%] of 31, for AP vs. bone vs. fat, respectively). Microsatellite DNA analysis confirmed that the eight cloned red deer calves were genetically identical to the cells used for NT.

New developments reproductive technologies in deer

In vitro embryo production is the platform for advanced reproductive technologies, such as cloning. The in vitro embryo production system developed for farmed red deer (Cervus elaphus) evolved along similar lines to that pioneered by other domestic species researchers. However, applying existing in vitro embryo production methods from these other species resulted in limited success and has necessitated developing a species-specific methodology for red deer based on the their physiology. Analysis of oviduct fluid led to the development of a semi-defined fertilization and culture media system, Deer Synthetic Oviduct Fluid (DSOF), which resulted in successful culture of red deer embryos to the blastocyst stage. Transvaginal ultrasound-guided ovarian examination and ovum pickup has enabled the study of seasonality constraint and propagation from selected female genetics, respectively. During the 4-month breeding season (April-July), 15% of cleaved oocytes developed to blastocysts, whereas no blastocysts developed from oocytes collected after July. The process of developing an in vitro embryo production system for farmed red deer may serve as a beneficial model for the propagation of endangered cervine species.

Embryo transfer in fallow deer ( Dama dama ): Superovulation, embryo recovery and laparoscopic transfer of fresh and cryopreserved embryos

Multiple ovulation-embryo transfer (MOET) protocols for farmed fallow deer (Dama dama) were investigated in a series of 3 experiments. A total of 37 donors, of either European (D.d. dama ; n = 30) or Mesopotamian hybrid (D.d. mesopotamica x D.d. dama ; n =7) genotype, each received an intravaginal silastic device containing 0.3 g progesterone (CIDR-type G device) for 14 d and injections of 0.5 units ovine FSH (8 x 0.06 unit injections from Days 10 to 14 of device insertion) and 100 IU PMSG (either with the first or last FSH injection). All donors received laparoscopic intrauterine inseminations of fresh semen (50 x 10(6) spermatozoa) from a Mesopotamian sire 36 h after withdrawal of CIDR devices. Embryos were recovered by laparotomy on Day 6 (Day 0 = estrus). Mean ovulation rates for the 3 experiments were 8.1, 9.8 and 7.0, with no effect of PMSG timing (P>0.10). However, embryo recovery rates, albeit low throughout the study (29.6%), were significantly improved with later PMSG administration (33.9 vs 20.1%; P<0.05). Hybrid and European donors performed in a similar manner. A range of embryo development stages was recovered throughout the study. In 2 experiments laparoscopic transfer of embryos to 48 recipient does treated previously with intravaginal CIDR devices for 14 d yielded a total pregnancy rate of 37.5%. In the experiment with fresh embryos, the use of clenbuterol to reduce uterine turgidity resulted in a higher proportion of does conceiving (3/4 ; 75%) compared with that of the untreated does (0/6 , 0%; P<0.05). In the second experiment, in which all the does routinely received clenbuterol, 10/19 (53%) and 5 19 (26%) does conceived following the transfer of fresh and cryopreserved embryos, respectively (P<0.05). While the overall efficiency of the MOET program was low (equivalent of 0.9 to 1.0 surrogate pregnancies per donor), improvements in the recovery rate of transferable embryos have considerable potential for genetic improvement of farm stock and captive propagation of endangered Mesopotamian fallow deer through maternal surrogacy programs.

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer ()

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10(6) sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green- and CT-Red-labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10(6)) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).

Specific Epiblast Loss and Hypoblast Impairment in Cattle Embryos Sensitized to Survival Signalling by Ubiquitous Overexpression of the Proapoptotic Gene BAD

Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies.

A mathematical model of the interaction between bovine blastocyst developmental stage and progesterone-stimulated uterine factors on differential embryonic development observed on Day 15 of gestation

A complex interaction between the developing bovine embryo and the growth potential of the uterine milieu it inhabits results in an embryo capable of developing past the maternal recognition stage and on to a successful pregnancy. Previously, we observed variation in the lengths of embryos recovered 8 d after bulk transfer of Day 7 in vitro-produced (IVP) blastocysts into the same uterus. Potential causes of the differential embryonic growth were examined and modeled using 2 rounds of bulk (n = 4-6) IVP transfers and recovery of these embryos 8 d later. Morphological and gene expression measurements of the embryos were determined and the progesterone concentration of the cows was measured throughout the reproductive cycle as a reflection of the status of the uterine environment. These data were used to develop and evaluate a model that describes the interaction between the uterine environment and the growth rate of the developing embryo. Expression of 6 trophectoderm genes (IFNT, TKDP1, PAG11, PTGS2, DKK1, and PDPN) was correlated with conceptus length. The model determined that if the embryo develops to blastocyst stage, the uterine environment, driven by progesterone, is a more important component than blastocyst size in the stimulation of embryonic growth rate to ensure adequate interferon tau (IFNT) for pregnancy recognition. We detected an effect of Day 7 progesterone on the expression of all 6 genes, embryonic disc size, and trophectoderm length on Day 15. We also found effects of embryo transfer size on trophectoderm length and expression of IFNT and PAG11 on Day 15. Lower energy balance over the period from transfer to recovery was associated with reduced embryo growth to Day 15, and this effect was independent of progesterone. Energy balance also affected expression of PDPN and TKDP1 on Day 15. We observed an effect of energy balance from transfer to recovery on embryo survival in cows with partial embryo losses, where embryo factors dominate embryo survival, with cows with greater energy balance having lower embryo losses. This effect was independent of energy balance 40 d before transfer and suggests that energy balance has direct, immediate effects on the embryo and maternal environment during this period. Furthermore, energy balance effects on embryo survival in cows with partial embryo losses were largely mediated by expression of TKDP1, PAG11, and PDPN. These results provide candidate signaling pathways for the effect of progesterone and energy balance on embryo growth and survival.