P.A. Pugh

Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer.

This study examined the effects of adding a macromolecule, polyvinylpyrrolidone (10% PVP) and a sugar (0.3 M trehalose) to vitrification solutions (VS) containing either one (40% ethylene glycol [EG], two (25% EG+25% DMSO) or three (20% EG+20% DMSO+10% 1, 3-butanediol [BD]) permeable cryoprotectants on the survival and hatching of IVP bovine embryos, following vitrification, warming and in-straw cryoprotectant dilution. Grade 1 and 2 compact morulae and blastocysts were selected on Day 7 (Day 0=IVF) of culture in SOFaaBSA and equilibrated for 10 min at room temperature in 10% EG. Following exposure, for up to 1 min at 4 degrees C, to one of the above VS (with or without PVP+trehalose), the embryos were loaded into straws and immersed in liquid nitrogen. Following warming and in-straw cryoprotectant dilution, the embryos were cultured for 48 h to assess hatching. There was no effect of VS on the survival of embryos after 24 h, however fewer compact morulae than blastocysts survived after 24 h (24% vs. 75%; P<0.001) or hatched after 48 h (15% vs. 59%; P<0.001). When blastocysts only were considered, an interaction between VS and additional PVP+trehalose was also observed (P<0.01). Hatching was reduced when they were added to 25% EG+25% DMSO (70% vs. 45%) but was not affected for either 40% EG (44 and 49%) or to 20% EG+20% DMSO+10% BD (72 and 72%). Pregnancy rates (Day 90 ultrasound) of recipients that were transferred either two non-vitrified or two vitrified (20% EG+20% DMSO+10% BD) blastocysts, did not differ (3/6 [50%] and 11/20 [55%]). However, significantly (P<0.02) fewer recipients that received compact morulae maintained pregnancy to Day 90 although this was not affected by vitrification (fresh vs. vitrified; 1/5 [20%] vs. 3/18 [17]). These data demonstrate that a VS comprising three cryoprotectants, rather than one, enables more embryos to hatch during post-thaw culture and that the survival, following direct transfer of these vitrified embryos, is not different to non-vitrified embryos.

Developmental ability of in vitro matured sheep oocytes collected during the nonbreeding season and fertilized in vitro with frozen ram semen

During the nonbreeding season, oocytes recovered from ovaries of FSH-primed or nonprimed ewes were matured in the presence or absence of granulosa cells collected from ovaries of primed or nonprimed ewes prior to in vitro fertilization with either fresh or frozen-thawed sperm. Following fertilization, ova were cultured for 24 h in synthetic oviduct fluid medium (SOF) supplemented with 20% human serum at 39 degrees C under humidified 5% CO(2), 5% O(2), 90% N(2) and then assessed for cleavage. Overall, 52% of ova cleaved. Cleavage was not affected by the source of sperm. Significantly more oocytes from primed follicles cleaved after 24 hours than those from nonprimed follicles (P<0.001). Maturation of oocytes in the presence of granulosa cells from nonprimed ewes resulted in a lower cleavage rate (44%, P<0.05) than in the presence of granulosa from primed ewes (59%) or no granulosa cells (50%). Oocytes (n = 508) from primed ewes were matured in the presence of granulosa cells (also from primed ewes) and fertilized in vitro with frozen-thawed sperm. Following in vitro culture for 24 hours, 68 of the 270 (53%) cleaved embryos were transferred to 17 recipient ewes, 15 of which remained pregnant to term, producing 24 lambs. The remaining 202 cleaved embryos were cultured for a further 5 days, of which 73 appeared to reach the morula/blastocyst stage and 61 were transferred to 16 recipients. Two ewes remained pregnant to term producing two lambs. These results demonstrate that production of sheep embryos using in vitro maturation and fertilization techniques is possible in the nonbreeding season. However, the poor viability of embryos obtained following extended culture needs to be resolved before such techniques can be usefully applied.

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.

In vitro development of early sheep embryos is superior in medium supplemented with human serum compared with sheep serum or human serum albumin

Abstract Two experiments were performed to assess the effect of supplemental protein source on sheep embryo development in vitro when incubated in synthetic oviduct fluid medium (SOF). In Experiment 1, a 2 × 3 factorial (N = 215) design examined two serum sources (20% v/v human serum (HS) vs. sheep serum (SS)) and three incubation vessels (multiwells, containing 500 μl media (MW); microwells, containing 50 μl media (μW); microdrops under paraffin oil, containing 50 μl media (μD)). One-cell embryos were incubated for approximately 144 h at 39°C under humidified 5% CO2, 5% O2, 90% N2. Incubation in medium containing HS resulted in more blastocysts (81 vs. 60%, P

Cryopreservation of in vitro-produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on post-thaw survival.

Two experiments were conducted to examine the effect of membrane stabilization through the modification of in vitro culture medium or freezing medium on post-thaw survival of in vitro-produced bovine embryos. In Experiment 1, Day 7 (Day 0 = day of IVF) late morulae and blastocysts that developed following culture in SOF/aa/BSA (IVC medium) were frozen slowly to -35 degrees C in the presence of 1.5 M ethylene glycol prepared in ovum culture medium (OCM) or in OCM supplemented with 10, 25 or 50% fetal calf serum (FCS) or 5, 10 or 25 mg/mL BSA. Post-thaw survival was assessed by re-expansion and/or hatching following 48 h of culture in IVC medium + 10% FCS. Overall, survival was significantly (P < 0.01) affected by embryo stage, with more hatched blastocysts surviving (71%) than blastocysts (59%) or late morulae (51%). Addition of FCS significantly (P < 0.01) reduced survival compared with control embryos or those frozen in BSA-supplemented medium (50.48 vs 68.01 vs 63.53%, respectively). There was also a significant interaction between embryo stage and protein type (P < 0.05). The survival of late morulae/early blastocysts following freezing was improved in the presence of additional BSA but not FCS. In Experiment 2, the IVC medium was supplemented with liposomes containing lecithin, sphingomyelin and cholesterol. Sphingomyelin and cholesterol at ratios of 1:1, 1:4 and 4:1 were added to 50, 100 or 150 micrograms/mL lecithin to yield a final lipid concentration of 200 micrograms/mL. A further group contained 200 micrograms/mL lecithin only. Blastocysts were frozen in 1.5 M ethylene glycol in OCM, then thawed and assessed as in Experiment 1. The presence of liposomes during IVC did not affect the proportion of cleaved embryos that developed to blastocysts or survival following freezing. However, the survival of blastocysts that developed in the presence of 200 micrograms/mL lecithin only was significantly lower than in any other treatment (6%; P < 0.03). These studies demonstrate that the protein composition of the freezing medium can significantly affect survival after thawing and that the survival of late morulae can be improved with additional BSA. The presence of lecithin only in the liposome preparation did not affect embryo development, but significantly reduced survival after freezing, suggesting it can affect post-thaw embryo survival, perhaps by altering embryonic membrane composition.

Development of in vitro embryo production systems for red deer (Cervus elaphus) Part 3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves

The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer (Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24h in TCM-199 supplemented with 10% foetal calf serum, 10 microg ml(-1) FSH and LH, and 1microg ml(-1) of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5mM Ca(2+) and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml(-1)) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present (P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner (P<0.001) and both approached an asymptote at 0.4 x 10(6) sperm ml(-1) with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations (P<0.05) but was variable (range 3.5+/-4.2 to 42.3+/-10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8-10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA (n=10) or on red deer epithelial oviduct monolayers (n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.

Effect of 2,4-dinitrophenol on the energy metabolism of cattle embryos produced by in vitro fertilization and culture

In cattle embryos, the proportion of ATP produced by glycolysis increases following the major activation of the embryonic genome, and development to the blastocyst stage is improved in the presence of 10 microM 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, from Day 5 to Day 7 of culture. In Experiment 1 of the present study, culture of cattle embryos in the presence of 10 microM DNP from Day 5 to Day 7 stimulated development to the blastocyst stage, but had no significant effects on oxygen, pyruvate or glucose uptake, or on lactate production. In Experiment 2, culture of cattle embryos in the presence of 10 microM DNP from Day 5 to Day 7, stimulated the metabolism of [2-14C]pyruvate (a measure of Krebs cycle activity) on all of Days 5, 6 and 7, and stimulated metabolism of [5-3H]glucose (a measure of glycolysis) on Day 7 only. The results show that 10 microM DNP stimulates oxidative and glycolytic metabolism in Day-5 to Day-7 cattle embryos, but this does not fully explain the observed increase in developmental competence. We propose that partial inhibition or uncoupling of oxidative phosphorylation may reduce the level of intracellular reactive oxygen species production, thereby facilitating development.

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.

In vivo survival of transferred sheep embryos following puncture of the zona pellucida and in vitro culture

This experiment (a 2×2×2 factorial design) investigated the effects on 1- and 2-cell sheep embryos of puncture (ZD) of the zona pellucida, as used in embryo cloning, subsequent in vitro culture for 3 or 5 days in synthetic oviduct fluid (SOF) plus 20% human serum, and transfer of these embryos to the uterus or oviduct of synchronised recipients (three embryos/recipient) on their subsequent development to recipient slaughter at Days 14 or 19. There was no effect of ZD on in vitro development to beyond the 8-cell stage (95%) after 3 days of culture or to blastocysts (74%) after 5 days of culture. However, survival after transfer of embryos cultured for 3 days (41%) was lower (P<0.01) than that of embryos cultured for 5 days (64%) and this was influenced by an interaction between site of transfer and culture length (P<0.001). Overall the survival after transfer of ZD embryos was lower than that of intact embryos (42% vs. 55%, P<0.05) and an interaction was observed between ZD and culture length (P<0.05). ZD embryos cultured for 3 days were less viable than intact embryos cultured for 3 days or embryos cultured for 5 days, indicating that the zona pellucida may play a protective role during early embryo development. The results demonstrate that the site of transfer of cultured embryos influences their subsequent survival; embryos cultured for 3 days should be transferred to the oviduct and 5-day cultured embryos should be transferred to the uterus. Culture of embryos to post-compaction stages in a system which supports high levels of development and subsequent in vivo survival may be useful for the selection of manipulated embryos for transfer.