Inclusion of bovine lipoproteins and the vitamin E analogue, Trolox, during in vitro culture of bovine embryos changes both embryo and fetal development.

This experiment investigated effects of lipoproteins and Trolox (vitamin E analogue) on bovine embryo and fetal development. The treatments were: in vitro culture (IVC) in synthetic oviducal fluid alone (SOF); with bovine lipoproteins (2% v/v; SOFLP); with Trolox (100µM; SOFT); and with lipoproteins and Trolox (SOFLPT). In vitro culture with lipoproteins increased fatty acid content of blastocysts (P<0.001) whereas inclusion of Trolox had no effect (P>0.05). Whereas lipoproteins reduced zygote development to blastocysts (P=0.03), Trolox facilitated increased development (P<0.001) and counteracted the reduction observed with lipoproteins (interaction, P=0.009). Lipoproteins also compromised (P<0.001) but presence of Trolox (P>0.05) had no effect on blastocyst morphological grade. Pregnancy rates resulting from synchronous transfer of IVP embryos were not affected by IVC treatment. At Day 70 of pregnancy, compared with SOF, fetal weight was lower in SOFLP but not SOFLPT (interaction, P<0.001). Liver weight (g kg(-1) fetal weight) was greater (P=0.03) in treatments containing Trolox. Placentome numbers were greater in SOF and SOFLPT compared with SOFLP and SOFT (interaction, P=0.002); superior embryo grades were also associated with increased numbers of placentomes (P=0.024). In conclusion, the interactive effects of lipoprotein and Trolox inclusion on in vitro embryo development were also evident in fetal development at Day 70.

Traversing the ovine cervix - a challenge for cryopreserved semen and creative science.

This review brings together research findings on cervical relaxation in the ewe and its pharmacological stimulation for enhancement of the penetration needed for transcervical insemination and embryo transfer. On the basis that the success of artificial insemination is the percentage of ewes lambing, a review is made of recent research aimed at understanding and minimising the sub-lethal effects of freezing and thawing on the viability of spermatozoa, their membrane integrity and their ability to migrate through cervical mucus, as these characteristics have a major influence on fertility, particularly when semen is deposited, artificially, in the os cervix. Milestones of achievement are given for transcervical intrauterine insemination, embryo recovery and transfer and the birth of lambs of pre-determined sex, firstly following intracytoplasmic sperm injection, then laparoscopic intrauterine insemination using highly diluted flow-cytometrically sorted fresh semen and subsequently by os cervix insemination using sexed semen that had been frozen and thawed. Diversity of research endeavour (applied, cellular, molecular), research discipline (anatomy, histology, immunology, endocrinology) and research focus (cell, tissue, organ, whole animal) is embraced within the review as each has significant contributions to make in advancing recent scientific findings from the laboratory into robust on-farm transcervical insemination and embryo transfer techniques.

Role of fatty acids in energy provision during oocyte maturation and early embryo development.

While much is known about the metabolism of exogenous nutrients such as glucose, lactate, pyruvate, amino acids by oocytes and pre-implantation mammalian embryos, the role of endogenous stores, particularly lipid, has been largely overlooked. The presence of lipid within oocytes and early embryos has been long known, and comparisons between species indicate that the amounts and types of lipid present vary considerably. Large amounts of intracellular lipid can compromise the success of cryopreservation and the removal of such lipid has been the subject of considerable effort. In this review, we present evidence that strongly suggests a metabolic role for lipid, specifically with regard to energy provision, in the late-stage oocyte and the pre-implantation embryo. We focus initially on oxygen consumption as a global indicator of metabolic activity, before reviewing different approaches that either have been designed to investigate directly, or have revealed indirectly the role of endogenous lipid in energy generation. These fall under five headings: (i) fatty acid oxidation; (ii) inhibition of triglyceride oxidation; (iii) culture in the absence of exogenous substrates; (iv) cytoplasmic organization; and (v) delipidation. On the basis of the data derived from these studies, we conclude that there is strong evidence for the utilization of endogenous lipid as an energy substrate by oocytes and early embryos.

Dietary carbohydrates and amino acids influence oocyte quality in dairy heifers.

The objective of the present experiment was to determine whether increasing plasma insulin by different nutritional regimes affects oocyte quality. Holstein dairy heifers (eight per treatment) were assigned, using a two times two factorial design, to diets containing either low or high dietary leucine and either low or high dietary starch. Each heifer underwent six sessions of ovum pick-up beginning 25 days after introduction of the diets. Oocyte quality was assessed by development to the blastocyst stage in synthetic oviducal fluid following in vitro fertilisation. Feeding diets containing high leucine resulted in significantly higher plasma free leucine and tyrosine concentrations. The high-starch diet significantly increased plasma insulin but not glucagon concentration, whereas high dietary leucine increased plasma glucagon but not insulin. Oocyte cleavage was not influenced by diet. The high-starch diet, which was associated with a high plasma insulin : glucagon ratio, had adverse effects on oocyte quality that were avoided when leucine intake was increased. There was an association between total plasma free amino acid concentration and oocyte cleavage. Therefore, in dairy heifers dietary amino acids and carbohydrates during antral follicle development appear to mediate effects on oocyte quality by different mechanisms. These findings have implications for both diet formulation and feeding regimes.

Feeding frequency has diet-dependent effects on plasma hormone concentrations but does not affect oocyte quality in dairy heifers fed fibre- or starch-based diets.

The post-fertilisation developmental capacity of bovine oocytes recovered by ultrasound guided transvaginal follicular aspiration (ovum pick-up, OPU) is influenced by diet-induced changes in hormone and metabolite concentrations. The objectives of this experiment were first to determine whether post-prandial changes in hormone concentrations, induced by changing the frequency of feeding, influenced oocyte quality and second whether changes in plasma glucagon concentration were associated with oocyte quality. Using a 2 × 2 factorial design, Holstein heifers (six per treatment) were fed either fibre- or starch-based diets containing either 189 or 478 g starch/kg dry matter. The diets were offered in either two or four equal meals per day and supplied twice the maintenance energy requirement. Blood samples were obtained both at weekly intervals (three samples per heifer, collected before feeding) during the experiment and throughout an entire 24-h period (15 or 17 samples per heifer for twice or four times daily-fed heifers, respectively). Each heifer underwent six sessions of OPU (twice weekly) beginning 25 days after introduction of the diets. Oocyte quality was assessed by development to the blastocyst stage in synthetic oviductal fluid following in vitro fertilisation. Mean weekly plasma insulin concentrations did not differ between diets, but plasma glucagon concentrations were greatest when heifers were fed the starch-based diet twice daily compared with the other diets. When heifers were offered four meals per day, there were no meal-related changes in hormone concentrations. However, when heifers were offered two meals per day, plasma insulin concentration increased after feeding the starch-based, but not the fibre-based diet. Plasma glucagon concentration increased after meals when heifers were fed twice daily and the increase was substantially greater when the starch-based diet was fed. Treatments did not influence (overall mean with mean ± s.e.) ovarian follicle size distribution or oocyte recovery by OPU (6.2 ± 0.4 per heifer), the proportion of oocytes that cleaved following insemination (0.57 ± 0.030) or blastocyst yield (0.27 ± 0.027 of oocytes cleaved). In conclusion, by feeding diets differing in carbohydrate source at different frequencies of feeding, meal-related changes in plasma hormone profiles were altered significantly, but oocyte quality was not affected. Therefore effects of diet on oocyte quality appear not to be mediated by meal-related fluctuations in hormone concentrations.

Ovine fetal development is more sensitive to perturbation by the presence of serum in embryo culture before rather than after compaction.

The effects on subsequent fetal development of the presence or absence of serum at different times during IVC of ovine zygotes were studied. Zygotes, recovered from superovulated ewes 36h after intrauterine AI using semen from a single sire, were cultured for 5 days in synthetic oviductal fluid (SOF) media supplemented with either BSA and amino acids (SOF-) or with 10% (v/v) steer serum (SOF+). Serum was present or absent during the first two and last 2 days of IVC giving four treatments (SOF-/SOF-; SOF-/SOF+;SOF+/SOF- and SOF+/SOF+). In total, 224 embryos, including 26 in vivo controls, were transferred singly at day 6 post-AI to synchronous recipients and the products of conception recovered at day 125 of gestation. Presence of serum during IVC had a biphasic effect on embryo development. The inclusion of serum during the first 2 days of IVC retarded early embryo development while the inclusion of serum during the last 2 days of IVC produced more blastocysts by day 6. These effects were independent of each other. The presence of serum during the first 2 days of IVC resulted in increased weights of gravid uterus, placenta, fetus, fetal heart and liver. The incidence of fetuses whose total or organ weights were greater than three standard deviations above the corresponding mean weights of control fetuses was also greater when serum was present during the first 2 days of IVC. However, even when serum was absent throughout IVC there was still an infrequent incidence of fetal weights greater than three standard deviations above the mean for control fetuses. These observations provide evidence that it is the early pre-compaction stages of embryo development that are particularly sensitive to perturbations leading to abnormal fetal development.

Zygote donor nitrogen metabolism and in vitro embryo culture perturbs in utero development and IGF2R expression in ovine fetal tissues.

Tests were made of the effects of altering nitrogen metabolism in zygote donor ewes on fetal development and expression of the gene encoding the type II insulin-like growth factor receptor (IGF2R) following the transfer of ovine embryos cultured from these zygotes, either in the absence or presence of serum. Zygotes, recovered from superovulated ewes (32 on a urea supplemented (30 g urea/kg) diet (high N) and 32 on a control diet (low N)) 36 h after intrauterine AI using semen from a single sire, were cultured for 5 days in synthetic oviductal fluid (SOF) media either with BSA and amino acids (SOF-) or with 10% (v/v) steer serum (SOF+). In total, 166 embryos, including 30 in vivo controls, were transferred singly at day 6 post-AI to synchronous recipients and the products of conception recovered at day 125 of gestation. Elevated plasma urea concentrations in zygote donors were associated with accelerated early embryo development, low pregnancy rates (16%) for embryos from the high N, SOF+ treatment, and significantly influenced fetal development and the expression of IGF2R in the fetal heart at day 125 of gestation. Importantly, the culture of sheep zygotes under serum-free conditions led to a high incidence of aberrant conceptus development and IGF2R expression. Consequently, maternal nitrogen metabolism prior to zygote recovery and in vitro culture can influence fetal development and the expression of an imprinted gene following embryo transfer, and these data support the notion that environmental effects on the follicle-enclosed oocyte may contribute to the etiology of the Large Offspring Syndrome.

Embryo technologies and animal health - consequences for the animal following ovum pick-up, in vitro embryo production and somatic cell nuclear transfer.

Mammalian reproductive technologies that aim either to complement or to transcend conventional livestock breeding options have contributed to some of the most remarkable achievements in the field of reproductive biology in recent decades. In so doing they have extended our horizons in two distinct dimensions, the first concerning what it is technically possible to achieve and the second relating to the time-frame within which an individual's life-long developmental capability is initially established and ultimately realized or undermined. Our impressions of the benefits and values, or otherwise, of technologies such as in vitro embryo production and nuclear transfer are rightly influenced by the extent to which they impinge on the health of animals either subjected to or derived from them. Here, we consider some of the health implications of oocyte/embryo-centric technologies applied to farm livestock.

Accumulation and distribution of neutral lipid droplets is non-uniform in ovine blastocysts produced in vitro in either the presence or absence of serum.

Sheep zygotes were cultured in serum-free or serum-supplemented media to determine effects on blastocyst yields and within-blastocyst abundance and distribution of neutral lipid droplets. Embryos cultured in synthetic oviduct fluid supplemented with bovine serum albumin (0.4% w/v) (SBSA) generated similar blastocyst yields (mean +/- s.e.m. = 20% +/- 5) to those in synthetic oviduct fluid supplemented with serum (10% v/v) from ewes fed a diet containing 0% (SZFO; 26% +/- 2) or 3% fish oil (S3FO; 23% +/- 3). SBSA zygotes generated more good-quality blastocysts than their SZFO or S3FO counterparts (P < 0.05). Within-blastocyst abundance of neutral lipid droplets was non-uniform; data were collected from discrete embryo sectors (each = 2700 microm2) representing highest (H), intermediate (I) and lowest (L) densities of accumulation. For all sectors, area (microm2) occupied by lipid droplets in SBSA blastocysts (mean H = 470; I = 370; L = 245) was smaller (P < 0.01) than occupied in others (SBSA : SZFO = 1 : 1.41, 1 : 1.48 and 1 : 1.42; SBSA : S3FO = 1 : 1.36, 1 : 1.30 and 1 : 1.31; data for H, I and L, respectively). Among S3FO blastocysts only, inferior quality was associated with greater lipid abundance. Overall, embryo culture in the presence of serum increased neutral lipid droplet abundance but accumulation was non-uniform.

Effect of ammonium chloride on the growth and metabolism of bovine ovarian granulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride.

Three experiments determined first, the effect of increasing ammonium chloride (NH(4)Cl) concentrations on the growth and metabolism of bovine granulosa cells isolated from small and medium-sized bovine ovarian follicles; secondly, whether the changes in granulosa cell growth and metabolism induced by NH(4)Cl were reversible; and thirdly, whether granulosa cells, previously conditioned with NH(4)Cl, were able to support maturation of oocytes in vitro. In Experiment 1, using a 2 (follicle size class) x 5 (NH(4)Cl concentration) factorial design, granulosa cells from small or medium-sized ovarian follicles were incubated for 96 h with 0, 0.2, 0.4, 0.8 or 1.6 micromol NH(4)Cl/ml. Experiment 2 used a split plot factorial design where granulosa cells were incubated for 96 h in the presence or absence of 1 micromol/ml NH(4)Cl and then incubated in the absence or presence of 1 micromol/ml NH(4)Cl for a further 48 h. Finally in Experiment 3, ovine oocytes were matured on layers of bovine granulosa cells which had not been conditioned with NH(4)Cl or conditioned with 0.5 or 1.0 micromol/ml NH(4)Cl and development of embryos to the blastocyst stage followed and blastocyst quality assessed. In Experiment 1, incubation of granulosa cells in increasing concentrations of NH(4)Cl reduced cell growth, increased cell protein concentrations and increased the amounts of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) oxidised and oestradiol and progesterone produced per 10(5) cells. Cells from medium-sized follicles were more sensitive to NH(4)Cl concentration and oxidised more MTT and produced less progesterone at high NH(4)Cl concentrations than cells from small-sized follicles. When, in Experiment 2, NH(4)Cl was removed from cell culture after 96 h incubation, cells previously exposed to NH(4)Cl grew at a slower rate during the subsequent 48 h, contained more cellular protein, oxidised more MTT and produced more oestradiol and progesterone than cells not previously exposed to NH(4)Cl. Maturation of ovine oocytes in coculture with bovine granulosa cells not exposed to NH(4)Cl (Experiment 3) increased egg cleavage rate and the proportion of cleaved eggs which developed to the blastocyst stage. Conditioning of granulosa cells with NH(4)Cl supported egg cleavage and development to the blastocyst stage at rates similar to those observed in the absence of granulosa cells. In conclusion, these experiments showed that the in vitro growth and metabolism of granulosa cells were altered by concentrations of NH(4)Cl similar to ammonium ion concentrations measured in follicular fluid and that these effects were not immediately reversible. Furthermore, the ability of granulosa cells conditioned with NH(4)Cl to support in vitro maturation of oocytes was impaired.

Consequences of manipulating gametes and embryos of ruminant species.

During the past 12 years, ruminants have provided a focus for some significant advances in mammalian reproductive biotechnologies. Lambs were the first offspring generated after nuclear transfer of fetal or adult cells to enucleated oocytes, and many calves of pre-determined gender are today the result of commercialized semen sexing. In 1990, the birth of one calf provided living proof that even 'dead' spermatozoa can be paternal, whereas, more recently, a short-lived gaur calf and viable mouflon lamb represented a novel option for conservation of endangered species. As well as highlights, hazards have emerged, resulting in setbacks or developmental anomalies, such as those associated with the large offspring syndrome which encompasses a range of adverse fetal, placental and post-natal phenomena expressed in ruminants. In this review, the developmental and other consequences of applying manipulative procedures, such as assisted fertilization, semen sexing, cloning and gene transfer, to gametes and embryos from bovine, ovine and caprine species are considered. Although assisted fertilization techniques can overcome mammalian infertility, they also usurp natural gamete selection safeguards, but not always with impunity. In the case of manipulations such as cloning, and to a lesser extent gene transfer, it is evident that nuclear-cytoplasmic interactions and nuclear-mitochondrial DNA interdependences are at least partially damaged or destroyed with a view to reconstruction. Therefore, among surviving zygotes and embryos it is inevitable that the legacy is frequently one of altered genetic, epigenetic or cellular programmes and processes.

Regulation of nutrient uptake and metabolism in pre-elongation ruminant embryos.

Our current understanding of pre-elongation embryo metabolism and its regulation by factors both intrinsic to the embryo and present in its immediate environment is limited mainly to studies in rodents and of ruminant embryos that have been cultured in vitro. Energy metabolism in such embryos is initially low and dependent on oxidative phosphorylation for the generation of ATP. The embryo exhibits substrate preference for carboxylic acids, such as pyruvate, during this period. Glucose uptake is limited initially but increases after compaction, and it is metabolized mostly to lactate. Glucose uptake is facilitated by a number of transporters, but the presence and function of these, and their regulation by growth factors, such as insulin, are not well characterized. Even less is known about the metabolic fate of amino acids and lipids. Approximately 50% of the lipid fraction in the mature oocyte is in the form of triglyceride, much of which is oxidized during fertilization and the early cleavage stages. Immunoreactivity of the growth hormone receptor is detectable from day 3 after fertilization, and so growth hormone acting in either a paracrine or endocrine manner may serve to regulate glucose, glycogen and lipid metabolism. The metabolic and mitogenic actions of insulin and insulin-like growth factor (IGF) I and II are thought to be mediated mainly by the IGF-I receptor. The actions of leptin in the ruminant embryo are less well understood. Circulating concentrations of these growth factors, together with nutrients supplied to the follicle and oviduct, can be modified by diet, but in ways that are not fully understood. The present review discusses these issues and highlights areas for future research endeavour where emphasis is directed on to combining thoughtfully designed whole animal studies with in vitro culture experiments.

Consequences of exposure to serum, with or without vitamin E supplementation, in terms of the fatty acid content and viability of bovine blastocysts produced in vitro.

To determine whether serum supplementation influenced fatty acid content of bovine blastocysts and whether vitamin E addition to culture medium containing serum could improve development in vitro, cleaved eggs were cultured in synthetic oviduct fluid supplemented with bovine serum albumin (BSA, 0.4% w/v, fraction V) (SVBSA), fetal calf serum (FCS, 10% v/v) (SFCS) or FCS (10% v/v) plus 100 micro M vitamin E (SFCS + E). Blastocyst yields were recorded and fatty acid composition was determined by gas chromatography. Day 7 blastocysts were incubated with [2-(14)C] pyruvate for 3 h and then fixed for cell counts. Yields of good quality blastocysts were greatest from cleaved eggs cultured in serum-free conditions (P < 0.01). In the presence of serum, supplementation with vitamin E increased both total and good quality blastocyst yields (P < 0.01). Presence of serum increased fatty acid content (mean +/- SEM) of blastocysts (SVBSA v. SFCS = 57 +/- 2 v. 74 +/- 2 ng embryo(-1); P < 0.001). In contrast, pyruvate metabolism was greater in blastocysts produced without serum (27 +/- 3 v. 21 +/- 3 picomoles embryo(-1) 3h(-1); P < 0.01) but, on a per cell basis, no differences were detected. Addition of vitamin E to the serum-supplemented formulation did not alter either the fatty acid content (73 +/- 2 ng embryo(-1)) or pyruvate metabolism index (19 +/- 1 pmol embryo(-1) 3h(-1)) of SFCS + E blastocysts. Thus, despite lipid accumulation, supplementary vitamin E improved blastocyst yields in embryos exposed to serum.

Manipulation of domestic animal embryos and implications for development.

Assisted reproductive technologies, as applied to domestic animals, can exert both novel and wide-ranging influences on the development, viability and welfare of offspring. Some of the changes are evident immediately or soon after the time at which a manipulative procedure is carried out, while other changes may not be evident until later in development or, perhaps, may remain undetected throughout an animal's lifetime. The present review explores some of the consequences - in terms of foetal, placental, neonatal and post-natal effects - of exposing embryos of cattle, sheep and other species to in vitro culture per se or, during culture, to physically invasive technologies including gene injection and nuclear transfer. The innate sensitivity of oocytes and recently fertilized eggs to their in vitro environment is illustrated by an examination of the later developmental repercussions resulting from apparently innocuous choices related to in vitro culture medium formulations. In contrast, an inherent resilience and paradoxical readiness to resume development following the traumas of nuclear transfer procedures is also in evidence. The extent to which assisted reproductive technologies will succeed, where relevant, in the domestic animal sector will be influenced by our appreciation of embryo requirements, for both short- and long-term developmental fitness, during their earliest developmental stages. Evidence of species-specific needs is testimony to the challenges ahead. Ultimately, our ability and inclination to resolve the limitations associated with current procedures will probably be greatly enhanced if predictive indicators (genetic, epigenetic or functional markers) of later developmental fitness can be identified.

Effects of epidural injections and transvaginal aspiration of ovarian follicles in heifers used repeatedly for ultrasound-guided retrieval of ova and embryo production.

Postmortem examinations of 13 Simmental heifers that had received between 16 and 28 injections to induce caudal epidural anaesthesia, the last not less than seven months before they were slaughtered, showed that none of them had any evidence of infection or inflammation at the injection site or in adjacent bone and soft tissues. Seven of them had minor damage to intercoccygeal discs, consisting of discospondylosis with neovascularisation and chondroid metaplasia, consistent with injuries caused by needles. The severity of the damage was not related to the number of epidural injections received, suggesting that the damage was probably caused by a discrete suboptimal injection procedure. In a second study, the ovaries from 22 Simmental heifers that had undergone between 13 and 16 transvaginal follicular aspirations were examined postmortem. Approximately one-third of them had a natural texture with little or no evidence of scar tissue, and less than one in five had extensive scarring and a toughened texture. There was no evidence of compromised ovarian function, as determined by the number and normality of corpora lutea and large follicles, in any of the animals.

Season affects characteristics of the pre-ovulatory LH surge and embryo viability in superovulated ewes.

The aim of this study was to determine whether there are seasonal shifts in ovulatory response, and in the viability of ova recovered from superovulated ewes. Fifty mature ewes underwent a standard oestrous synchronisation (CIDR), superovulation (oFSH) and artificial insemination procedure during October (peak breeding season) and April (transition to anoestrus). In each month peripheral LH and progesterone concentrations were measured around the time of ovulation and embryos were recovered, graded and cryopreserved on day 6 after insemination. During the subsequent breeding season, grade 1 and 2 morulae and unexpanded blastocysts were thawed and transferred singly to synchronous recipients (October, n = 40; April, n = 40) or cultured in vitro for 18-20 h (October, n = 107; April, n = 98). Following culture, viable embryos were stained to count cell nuclei or assayed to measure their capacity for glucose metabolism ([3H]glucose) and protein synthesis ([35S]methionine). Peak LH concentrations were higher in October than in April (38.2 +/- 3.26 ng ml(-1) versus 25.7 +/- 1.99 ng ml(-1), respectively; P < 0.01) and the pre-ovulatory LH surge was advanced by approximately 3 h (P < 0.05). Progesterone concentrations at CIDR withdrawal were lower in October than in April (3.1 +/- 0.16 ng ml(-1) versus 4.3 +/- 0.19 ng ml(-1), respectively; P < 0.001) but were not different at embryo recovery. Season did not affect the numbers of corpora lutea per ewe or the numbers of ova recovered but the proportion of recovered ova that was unfertilised/degenerate was lower in October than in April (0.43 versus 0.58, respectively; P < 0.001). For embryos containing more than 16 cells, there was no effect of season on the median stage of development or morphological grade. The proportions of October and April embryos that established pregnancy following transfer to recipient ewes were 0.78 and 0.70 (not significantly different), and that were viable after in vitro culture were 0.66 and 0.37 (P < 0.05), respectively. Season did not affect the number of nuclei per viable embryo or the capacity for protein synthesis but the glucose uptake of October embryos was approximately double that of April embryos (3163+/-293.4 dpm versus 1550+/-358.9 dpm, respectively; P < 0.05). Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilisation failure as a possible consequence of seasonal shifts in LH secretion and (or) associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo but those produced during the late, as opposed to the peak breeding season have lower viability following in vitro culture.

Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers.

To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCC's) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals. More OCC's were collected during OPU1 than OPU2 (means +/- S.E.M. = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%). The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M. = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively). Corresponding blastocyst yields from good quality OCC's (24 +/- 3% and 26 +/- 4%) also did not differ. Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05). In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1. Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCC's suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones. Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process.

Development, amino acid utilization and cell allocation in bovine embryos after in vitro production in contrasting culture systems.

The effects of protein-supplemented and protein-free media on amino acid uptake, protein synthesis and cell differentiation in bovine blastocysts were investigated. Four formulations of synthetic oviduct fluid were used. Each formulation was identified by the principal supplement: bovine serum albumin (0.4%, w/v); polyvinyl alcohol (0.3%, w/v); or either of two steer sera (10%, v/v). After zygote culture, blastocyst yields (day 7.5) were lowest in protein-free medium and highest in albumin-supplemented medium. Subsequent 12 h incubation in the presence of both essential and non-essential amino acids was used for the measurement of amino acid flux. All blastocysts released alanine but consumed aspartate (P < 0.001) and the extent was influenced by prior culture conditions. Aspartate uptake was lower in blastocysts produced in protein-free conditions (P < 0.05) than in blastocysts produced in albumin-supplemented conditions. Consumption indices for 16 other amino acids were not influenced by blastocyst source. Cell counts and hatching incidences were highest for albumin-supplemented blastocysts, but were similar among blastocysts from the protein-free and serum-dependent treatments. Crucially, the use of protein-free medium for zygote culture did not compromise resultant blastocysts in terms of either de novo protein synthesis ([3H]phenylalanine incorporation) or trophectoderm function (phenotype based on interferon-tau detection). Thus, although blastocyst yields were compromised after zygote culture in a protein-free (vis-à-vis albumin-supplemented) medium, amino acid flux was qualitatively conserved, and only quantitatively modified in the case of alanine and aspartate. Moreover, vital properties of blastocysts that were produced, including de novo protein synthesis and trophectodermal cell function, apparently were not adversely affected by protein deprivation.