M.P. Boland

Bovine Embryo Culture in the Presence or Absence of Serum: Implications for Blastocyst Development, Cryotolerance, and Messenger RNA Expression

Abstract We have previously shown that, while the intrinsic quality of the oocyte is the main factor affecting blastocyst yield during bovine embryo development in vitro, the main factor affecting the quality of the blastocyst is the postfertilization culture conditions. Therefore, any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the period of embryo culture in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and gene expression patterns. The quantification of all gene transcripts was carried out by real-time quantitative reverse transcription-polymerase chain reaction. Bovine blastocysts from four sources were used: 1) in vitro culture in synthetic oviduct fluid (SOF) supplemented with 3 mg/ml BSA and 10% fetal calf serum (FCS), 2) in vitro culture in SOF + 3 mg/ml BSA in the absence of serum, 3) in vitro culture in SOF + 16 mg/ml BSA in the absence of serum, and 4) in vivo blastocysts. There was no difference in overall blastocyst yield at Day 9 between the groups. However, significantly more blastocysts were present by Day 6 in the presence of 10% serum (20.0%) compared with 3 mg/ml BSA (4.6%, P < 0.001) or 16 mg/ml BSA (11.6%, P < 0.01). By Day 7, however, this difference had disappeared. Following vitrification, there was no difference in survival between blastocysts produced in the presence of 16 mg/ml BSA or those produced in the presence of 10% FCS; the survival of both groups was significantly lower than the in vivo controls at all time points and in terms of hatching rate. In contrast, survival of blastocysts produced in SOF + 3 mg/ml BSA in the absence of serum was intermediate, with no difference remaining at 72 h when compared with in vivo embryos. Differences in relative mRNA abundance among the two groups of blastocysts analyzed were found for genes related to apoptosis (Bax), oxidative stress (MnSOD, CuZnSOD, and SOX), communication through gap junctions (Cx31 and Cx43), maternal recognition of pregnancy (IFN-τ), and differentiation and implantation (LIF and LR-β). The presence of serum during the culture period resulted in a significant increase in the level of expression of MnSOD, SOX, Bax, LIF, and LR-β. The level of expression of Cx31 and Cu/ZnSOD also tended to be increased, although the difference was not significant. In contrast, the level of expression of Cx43 and IFN-τ was decreased in the presence of serum. In conclusion, using a combination of measures of developmental competence (cleavage and blastocyst rates) and qualitative measures such as cryotolerance and relative mRNA abundance to give a more complete picture of the consequences of modifying medium composition on the embryo, we have shown that conditions of postfertilization culture, in particular, the presence of serum in the medium, can affect the speed of embryo development and the quality of the resulting blastocysts. The reduced cryotolerance of blastocysts generated in the presence of serum is accompanied by deviations in the relative abundance of developmentally important gene transcripts. Omission of serum during the postfertilization culture period can significantly improve the cryotolerance of the blastocysts to a level intermediate between serum-generated blastocysts and those derived in vivo. The challenge now is to try and bridge this gap.

Analysis of Differential Messenger RNA Expression Between Bovine Blastocysts Produced in Different Culture Systems: Implications for Blastocyst Quality

Abstract Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

Temporal Divergence in the Pattern of Messenger RNA Expression in Bovine Embryos Cultured from the Zygote to Blastocyst Stage In Vitro or In Vivo

Abstract The objective of this study was to examine the time during the postfertilization period that gene expression patterns in in vitro-cultured bovine embryos diverge from those of their in vivo-cultured counterparts. Presumptive bovine zygotes were produced by in vitro maturation and fertilization of immature oocytes collected from the ovaries of slaughtered animals. Approximately 20 h post insemination (hpi), zygotes were denuded and randomly divided into two groups for culture either in vitro, in synthetic oviduct fluid medium, or in vivo, in the ewe oviduct. Embryos were recovered from both systems at approximately 30 hpi (2-cell), 2 (4-cell), 3 (8-cell), 4 (16-cell), 5 (early morula), 6 (compact morula), or 7 (blastocyst) days post insemination. On recovery, they were examined for stage of development and snap frozen in liquid nitrogen for the analysis of transcript abundance using real-time polymerase chain reaction. The transcripts studied were glucose transporter 5, sarcosine oxidase, mitochondrial Mn-superoxide dismutase, connexin 43, interferon tau, insulin-like growth factor II, apoptosis regulator box-α and insulin-like growth factor-I receptor, most of which are known from our previous work to differ in terms of transcript abundance in blastocysts derived from culture in vitro or in vivo. The results demonstrate that the relative abundance of the transcripts studied varies throughout the preimplantation period and is strongly influenced by the culture environment. In addition, the data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident by as little as 10 h of initiation of culture. Such information has implications not only for basic biology but also for human assisted reproduction in which there is a move toward culturing embryos to the blastocyst stage, necessitating prolonged culture in vitro under potentially deleterious conditions.

EFFECT OF NUTRITION ON ENDOCRINE PARAMETERS, OVARIAN PHYSIOLOGY, AND OOCYTE AND EMBRYO DEVELOPMENT

Reproductive efficiency in high yielding dairy cows has decreased over the past 50 years, despite significant gains in genetic selection for increased milk output. One possible reason for this decline has been a change in the nutritional intake to meet the increased energy and protein demands for higher milk production. Excess energy intake in sheep will lead to significant reductions in progesterone concentrations; the effects in cattle are not so clear. Nutrition, unless radically changed, will have little effect on gonadotropin concentrations in ruminants, and this is in contrast to the situation for pigs and for primates, where very short-term nutritional changes manifest themselves in altered gonadotropin secretion. Cattle with reduced energy intake have smaller dominant follicles and more three-wave cycles, compared with animals on higher feed intakes. One of the main areas where nutrition influences reproductive efficiency is at the level of embryo production. Several studies indicate that excess energy intake reduces the response to superovulation and also decrease the yield of embryos and alters expression of some gene constructs within the developing embryo. The mechanism of this effect is not clear but indications are that the quality of the oocytes may be compromised. Indeed recent data indicate that nutritional changes around the time of mating may have detrimental effects on the establishment of pregnancy in heifers. Thus, nutritional balancing is critical for high-yielding dairy cows, in particular. The challenge remains to modify nutritional and management strategies in such cows to maintain the levels of production made possible by genetic selection and still maintain an acceptable level of fertility.

Progesterone-Regulated Changes in Endometrial Gene Expression Contribute to Advanced Conceptus Development in Cattle

The postovulatory rise in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our study objective was to determine how elevated P4 alters endometrial gene expression to advance conceptus development. Synchronized heifers were inseminated (Day 0) and randomly assigned to pregnant high P4 or to pregnant normal P4. All high P4 groups received a P4-release intravaginal device on Day 3 after insemination that increased P4 concentrations up to Day 7 (P < 0.05). Tissue was collected on Day 5, 7, 13, or 16 of pregnancy, and endometrial gene expression was analyzed using the bovine Affymetrix (Santa Clara, CA) microarrays. Microarray analyses demonstrated that the largest number of P4-regulated genes coincided with the day when the P4 profiles were different for the longest period. Genes with the largest fold change increase (such as DGAT2 and MSTN [also known as GDF8]) were associated with triglyceride synthesis and glucose transport, which can be utilized as an energy source for the developing embryo. Temporal changes occurred at different stages of early pregnancy, with the greatest difference occurring between well-separated stages of conceptus development. Validation of a number of genes by quantitative real-time PCR indicated that P4 supplementation advances endometrial gene expression by altering the time (FABP, DGAT2, and MSTN) or duration (CRYGS) of expression pattern for genes that contribute to the composition of histotroph.

Nutritional effects on ovulation, embryo development and the establishment of pregnancy in ruminants

The effects of high and low dietary dietary intake on reproduction in female cattle and sheep will be considered at the level of the pituitary gland, ovary and uterus. In sheep, increased dietary intake for a relatively short time will increase ovulation rate, by increasing gonadotropin secretion. Dietary intake can affect steroids such as progesterone and also intra-follicular concentrations of some growth factors such as IGF-1 and IGF-2. The effects of altered energy intake on gonadotropins and steroids in cattle are not as repeatable as those in sheep but follicular growth rates can be altered. High nutrition has a negative effect on oocyte quality, with animals on ad-libitum high energy diets particularly at risk. Overfeeding can decrease embryo quality in both sheep and cattle and it appears that this results from changes primarily at the level of the follicle or oocyte. Restricted nutrition for a short time will enhance pregnancy rates in cattle; most of this benefit appears to occur if food is restricted before insemination. Thus feeding levels before mating are particularly important to subsequent reproductive success. High dietary crude protein may decrease pregnancy rate in lactating cows. In ewes and heifers supplementation with urea failed to have any effect on pregnancy rates when good quality embryos were transferred to recipient animals exposed to high dietary crude protein. In donor ewes there were adverse effects on early embryo development following urea treatment, suggesting that the mechanism affecting the reproductive process was primarily operating at the level of the oocyte. Collectively, these data identify the overall deleterious effects of high dietary intake and excess crude protein on fertility and highlight the importance of dietary intake before ovulation on the likelihood of establishing a viable pregnancy.

Culture of in vitro produced bovine zygotes in vitro vs in vivo: Implications for early embryo development and quality

The objectives of this study were to examine the effect of culture system on bovine blastocyst formation rates and quality. Presumptive IVM/IVF bovine zygotes were cultured either in vitro in synthetic oviduct fluid (SOF, 25 embryos/25 microL in 5% CO2, 5% O2, 90% N2 at 39 degrees C) or in vivo in the ewe oviduct (approximately 100 embryos per oviduct). The recovery rate after in vivo culture was 53% (813/1,530). The blastocyst rate on Day 7 was significantly higher for the in vitro system (28%, 362/1,278 vs 17%, 37/813; P< 0.0001). However, after culture in vitro for a further 24 h, there was no difference in Day 8 yields (36%, 457/1,278 vs 32%, 258/813, for in vitro and in vivo culture, respectively). There was no difference in blastocyst cell number between treatments (Day 7: 96 vs 103; Day 8: 78 vs 85 for in vitro and in vivo culture, respectively). Irrespective of culture system, Day 7 blastocysts had a significantly higher cell number than those appearing on Day 8. There was no difference in pregnancy rate at Day 35 after fresh transfer of a single Day 7 blastocyst (37.5%, 21/56 vs 45.3%/, 24/53 for in vitro and in vivo culture, respectively). After cryopreservation by freezing in 10% glycerol, VS3a vitrification or solid surface vitrification, the survival of in vitro cultured embryos was significantly lower than survival of embryos cultured in the ewe oviduct or those produced by superovulation of donors. In conclusion, these findings demonstrate that while bovine zygotes cultured in vitro are capable of rates of development similar to those of their in vivo cultured counterparts (in terms of Day 8 blastocyst yield, cell number and early pregnancy rate), there are significant differences in embryo cryosurvival. This suggests that current in vitro culture systems need to be improved to optimize embryo quality and pregnancy rates.

Waves of follicle development during the estrous cycle in sheep.

The pattern of ovarian follicle development in maiden cyclic lambs was characterized using the definition of a follicle wave as the changes in the number of follicles among the days of the estrous cycle, as originally defined in cattle by Rajakoski in 1960. We also examined the steroid content relationships among follicles on Days 5 (Wave 1) and 14 (Waves 2 and 3) of the estrous cycle. In Experiment 1, the ovaries of 20 cyclic lambs (40 to 45 kg) were examined daily using transrectal ultrasonography for 1 or 2 estrous cycles (n = 31 cycles). The number of small (2 and 3 mm in diameter), medium (4 and 5 mm) and large (> or = 6 mm) follicles were aligned with the beginning and end of the average length estrous cycle and then compared among days. Identified follicles were defined as those that grew to > or = 4 mm and remained at > or = 3 mm for > or = 3 d. The number of identified follicles emerging (retrospectively identified at 2 or 3 mm) per ewe per day was also aligned with the average length estrous cycle. In Experiment 2, ewe lambs were ovariectomized on Day 5 (n = 6) or 14 (n = 5) of the estrous cycle, then follicle diameters and follicular fluid concentrations of estradiol and progesterone were compared among follicles. Data were analyzed by repeated measures ANOVA and compared among days using Fishers LSD. In Experiment 1, either 2 (n = 10 cycles), 3 (n = 20 cycles) or 4 (n = 1 cycle) periods of emergence of identified follicles occurred during individual cycles, with estrous cycle lengths of 15.6 +/- 1.6, 16.1 +/- 1.1 and 17 d respectively. In animals with 2 or 3 periods of emergence of identified follicles, the total number of small, medium and large follicles differed (P < 0.05) among days of the estrous cycle showing a wave-like pattern. In Experiment 2, a single follicle collected on each of Days 5 and 14 of the cycle (6.2 +/- 0.2 and 3.9 +/- 0.2 mm in diameter) had a higher (P < 0.05) concentration of follicular fluid estradiol (36.2 +/- 4.4 and 50.9 +/- 21.6 ng/mL) than other follicles collected on the same day (next largest follicle: 4.3 +/- 0.3 and 3.5 +/- 0.4 mm; 4.3 +/- 0.9 and 18.2 +/- 6.7 ng/mL estradiol). The results showed that 1) there was a synchronous emergence of follicles associated with fluctuations in the number and size of follicles during the estrous cycle; 2) within a wave there was a hierarchy among follicles for diameter and steroid content; 3) ovarian follicle growth in ewe lambs occurred in 2 or 3 organized waves during the estrous cycle.

Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows.

The effects of milk yield, body condition score (BCS) and lactation number on the number of oocytes recovered and blastocysts formed were studied following in vitro maturation, fertilization and culture of bovine oocytes collected from 48 high and 46 medium genetic merit dairy cows in their first and third lactation. The cows were slaughtered between 125 and 229 d post partum. Ovaries were recovered, and 2- to 10-mm follicles were aspirated. Cleavage rate and number of blastocysts were determined at 44 h and 7 d after insemination, respectively. Oocytes from high genetic merit cows formed fewer blastocysts and had lower cleavage and blastocyst formation rates than those from medium genetic merit cows (0.36 +/- 0.19, 70.4 and 6.8% vs 0.85 +/- 0.22, 77.4 and 11.4%, respectively). The effect of milk production was tested by grouping cows in their third lactation into high and low groups. There was no difference in number of oocytes recovered and subsequent development into blastocysts between the cows in the high milk production group (4559 to 5114 kg, n = 20) and cows in the low yield (3162 to 3972 kg, n = 20) group (6.9 +/- 1.34 vs 8.9 +/- 1.32, respectively). The effect of BCS was tested by grouping cows in their first or third lactation into high and low groups. Cleavage and blastocyst formation rates were greater for oocytes from cows with a high BCS (3.3 to 4.0, n = 20) than a low BCS (1.5 to 2.5, n = 20) (75.7 vs 61.9% and 9.9 vs 3.0%, respectively). Cows in the first lactation yielded fewer oocytes (5.7 +/- 1.24) than cows in the third lactation (7.8 +/- 0.79). Thus, the quality of oocytes probably contributes to reduced fertility, often evident in high genetic merit dairy cows.

Oocyte ultrastructure in bovine primordial to early tertiary follicles.

Abstract The aim of the present study was to describe in detail the changes occurring in the cytoplasmic ultrastructure of the bovine oocyte from the onset of growth in the primordial follicle until the completion of growth in the tertiary follicle. Bovine oocytes from primordial, primary, secondary and early to mid-antral follicles were processed and analysed by light and transmission electron microscopy. The primordial follicular oocyte was characterized by numerous coated pits on the oolemma and the accumulation of free and organelle-related smooth (SER) and rough (RER) endoplasmic reticulum, round mitochondria and Golgi complexes around the nucleus, which was located slightly off centre. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, elongated mitochondria and Golgi complexes. During the secondary follicular stage, formation of the zona pellucida, development of gap junctions between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and reduction in the number of coated pits on the oolemma were seen. In the tertiary follicular oocyte up to 100 μm in diameter, the number of Golgi complexes and lipid droplets increased and the organelles were dislocated to the deep cortical region. During the final growth of the oocyte up to >120 μm, the organelles were dislocated further to the peripheral region, the extent of the free SER and RER compartments were reduced, the number of individual cortical granules increased, hooded mitochondria became abundant and the perivitelline space developed. In conclusion, the growth of the bovine oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte specific structures such as the zona pellucida and cortical granules.