Associations between postpartum phenotypes, cow factors, genetic traits, and reproductive performance in seasonal-calving, pasture-based lactating dairy cows.

The objective of this study was to evaluate associations between corpus luteum (CL) status, uterine health, body condition score (BCS), metabolic status, parity, genetic merit for fertility traits, and reproductive performance in pasture-based dairy cows managed for seasonal reproduction. First- and second-lactation (n = 2,600) spring-calving dairy cows from 35 dairy farms located in Ireland were enrolled in the study. Farms were visited every 2 wk, and animals that were at wk 3 (range: 14-27 d in milk) and wk 7 (range: 42-55 d in milk) postpartum were examined. Body condition score was measured using a 1-to-5 scale in 0.25-point increments. Transrectal ultrasound examination was performed at wk 3 and 7 postpartum to determine presence or absence of CL and ultrasound reproductive tract score (scale of G1-G4). Blood samples were collected at each visit, and the concentrations of glucose, ß-hydroxybutyrate (BHB), and fatty acids (FA) were analyzed using enzymatic colorimetry. Animals were grouped into 3 BCS categories [low (≤2.5), target (2.75-3.25), and high (≥3.5)], 2 CL categories (present or absent), 2 uterine health status categories (normal or abnormal), and 3 metabolic status categories [good (high glucose, low FA and BHB), poor (low glucose, high FA and BHB), and moderate (all other combinations)]. Fisher's exact test was used to test for associations between variables and was supplemented by logistic regression. More cows with a CL at wk 7 were served during the first 21 d of the breeding period compared with cows without a CL. Cows classified as having a uterine score of G3 or G4 at wk 3 and 7 had lower odds of pregnancy establishment during the breeding period compared with animals with a uterine score of G1 or G2. Animals with low BCS at wk 7 had lower odds of pregnancy establishment than cows with a target BCS. Cows classified as having good metabolic status at both wk 3 and wk 7 had greater odds of pregnancy establishment during the first 21 d of the breeding season than those classified as having poor metabolic status. Overall, primiparous cows had greater reproductive performance than second-parity cows. Animals in the quartiles with the best predicted transmitting ability for survival and calving interval had better reproductive performance compared with animals in the other quartiles. Cows that had better genetic merit for fertility traits and good metabolic status, achieved target BCS, and had a favorable ultrasound reproductive tract score and a CL present at wk 7 postpartum had superior reproductive performance.

Effect of dietary n-3 polyunsaturated fatty acid supplementation and post-insemination plane of nutrition on systemic concentrations of metabolic analytes, progesterone, hepatic gene expression and embryo development and survival in beef heifers.

Nutrition, and particularly dietary energy intake, plays a fundamental role in reproductive function in cattle. There is some evidence that supplemental omega-3 dietary polyunsaturated fatty acids (n-3 PUFA) can exert positive effects on fertility. The objectives of this study were to evaluate the effect of dietary n-3 PUFA supplementation, post-insemination energy plane of nutrition and their interaction on embryo survival in cattle. Crossbred beef heifers (n = 185) were individually offered barley straw ad libitum and 6 kg DM of concentrate supplemented with either a rumen-protected source of saturated fatty acid (palmitic; control, CON) or a partially rumen-protected n-3 PUFA-enriched supplement (n-3 PUFA). Estrous was synchronised using two injections of PG administered at 11-d intervals and following artificial insemination (AI = Day 0) 179 heifers exhibiting oestrus were inseminated and assigned to one of two dietary treatments: (i) remain on their pre-insemination high dietary plane of nutrition (High) or (ii) restricted to 0.6 × estimated maintenance energy requirements (Low) in a 2 × 2 factorial design. The heifers were then maintained on their assigned diets until slaughter and embryo recovery on Day 16 (n = 92) or pregnancy diagnosis by ultrasound scanning at Day 30 post-AI (n = 87). Plasma concentrations of fatty acids, metabolites, insulin, progesterone (P4) and insulin-like growth factor 1 (IGF-1) were measured at appropriate intervals. Hepatic expression of mRNA for aldo-keto reductase (AKR1C), cytochrome P450 2C (CYP 2C) and cytochrome P450 3A (CYP 3A) was examined. The n-3 PUFA supplementation increased plasma n-3 PUFA concentration (P < 0.05) and reduced n-6: n-3 PUFA ratio (P < 0.05). Plasma IGF-1 was higher for n-3 PUFA relative to the CON (P < 0.05) and for High compared with Low plane of nutrition post-AI (P < 0.05) groups. A low plane of nutrition post-AI increased plasma concentrations of progesterone from Days 7-16 after insemination (P < 0.001) but reduced embryo length (P < 0.001). Supplementation with n-3 PUFA reduced and tended to reduce hepatic expression of CYP2C (P = 0.01) and CYP3A (P = 0.08), respectively. However, while dietary n-3 PUFA supplementation and an abrupt reduction in nutrient status following insemination elevated plasma concentrations of n-3 PUFA and mid and late phase P4, respectively, there was no effect of either PUFA supplementation or post-insemination plane of nutrition on embryo survival.

Dietary polyunsaturated fatty acid supplementation of young post-pubertal dairy bulls alters the fatty acid composition of seminal plasma and spermatozoa but has no effect on semen volume or sperm quality.

The aim of this study was to examine the effects of dietary supplementation with rumen protected n-6 or n-3 polyunsaturated fatty acids (PUFA) on the quantity and quality of semen from young post-pubertal dairy bulls. Pubertal Holstein-Friesian (n = 43) and Jersey (n = 7) bulls with a mean ± s.e.m. age and bodyweight of 420.1 ± 5.86 days and 382 ± 8.94 kg, respectively, were blocked on breed, weight, age and semen quality (based on the outcomes of two pre-trial ejaculates) and randomly assigned to one of three treatments: (i) a non-supplemented control (CTL, n = 15), (ii) rumen-protected safflower (SO, n = 15), (iii) rumen-protected n-3 PUFA-enriched fish oil (FO, n = 20). Bulls were fed their respective diets, ad libitum for 12 weeks; individual intakes were recorded using an electronic feeding system for the initial 6 weeks of the feeding period. Semen was collected via electro-ejaculation at weeks -2, -1, 0, 7, 10, 11 and 12 relative to the beginning of the trial period (week 0). On collection, semen volume, sperm concentration and progressive linear motility (PLM) were assessed. On weeks -2, -1, 0, 10, 11, 12, semen was packaged into 0.25 mL straws and frozen using a programmable freezer. On weeks -1, 7 and 11; a sub-sample of semen was separated into sperm and seminal plasma, by centrifugation and stored at - 20 °C until analysis of lipid composition. Semen from 10 bulls per treatment were used for post-thaw analysis at weeks 10, 11 and 12 (3 straws per ejaculate). Sperm motility was analysed by computer assisted semen analysis (CASA). In addition, membrane fluidity, acrosome reaction and oxidative stress were assessed using flow cytometry. Sperm from bulls fed SO had a 1.2 fold higher total n-6 PUFA content at week 11 compared to week -1 (P < 0.01) while bulls fed FO had a 1.3 fold higher total n-3 PUFA content, in sperm by week 11 (P < 0.01). There was no effect of diet on semen volume, concentration or PLM of sperm when assessed either immediately following collection or post-thawing. Membrane fluidity and oxidative stress of sperm were also not affected by diet. The percentage of sperm with intact-acrosomes was lower in CTL bulls compared to those fed SO (P < 0.01). In conclusion, while the lipid composition of semen was altered following dietary supplementation with either n-6 or n-3 based PUFA, this did not lead to measurable improvements in the quantity or quality of semen produced by young post-pubertal dairy bulls.

Effect of grass dry matter intake and fat supplementation on progesterone metabolism in lactating dairy cows.

Progesterone (P4) metabolism in dairy cattle can be manipulated by alterations in dry matter intake and diet composition. Our objectives were to determine the effects of grazing allowance and fat supplementation on P4 metabolism in lactating dairy cows. Forty mid- to late-lactation Holstein-Friesian dairy cows were used in a completely randomized block design, with a 2 × 2 factorial arrangement of treatments. Cows were assigned to receive 1 of 2 pasture allowances (ad libitum allowance [AL], 9.5 kg dry matter per day, or restricted allowance [R] 7 kg dry matter per day) and 1 of 2 fat supplementation treatments (750 g per day saturated fat [F] or no fat supplement [NF]). All cows received an additional 4 kg per day of concentrate. Grass dry matter intake (GDMI) was measured 5 wk after the initiation of dietary treatment. Cows were treated with prostaglandin F(2α) (PGF(2α)) to eliminate the endogenous source of P4, and two intravaginal progesterone-releasing devices (CIDR) were inserted into each cow for a period of 8 days. Regular blood samples were taken before and after the removal of the intravaginal progesterone-releasing devices, and analyzed for P4 concentrations. The half-life (t½) and metabolic clearance rate (MCR) of P4 was calculated for each cow. There was no effect of GDMI or fat supplementation on the t½ or MCR of P4. There was a tendency for an interaction between GDMI and fat supplementation on the t½ of P4; cows on the restricted-F diet tended to have a longer P4 t½ than cows on the ad libitum-F diet. It was concluded that greater alterations in GDMI than achieved in the current study are required to change P4 metabolism. A combination of fat supplementation and restricted feeding slows P4 clearance, which may have beneficial implications for fertility.

The effect of strategic supplementation with trans-10,cis-12 conjugated linoleic acid on the milk production, estrous cycle characteristics, and reproductive performance of lactating dairy cattle.

The objective was to determine the effects of a protected (lipid-encapsulated) conjugated linoleic acid (LE-CLA) supplement on milk production, estrous cycle characteristics, and reproductive performance in lactating dairy cows on a pasture-based diet. Spring calving dairy cows (n=409) on a single pasture-based commercial dairy farm were used in a completely randomized block design. Cows were assigned to 1 of 2 dietary supplements [LE-CLA (n=203) or no supplement (control, n=206)]. The LE-CLA cows received 51 g/d of a lipid supplement containing 5 g of both trans-10,cis-12 and cis-9,trans-11 CLA from 0 to 60 d in milk. Milk samples were collected 3 times weekly, and each sample was analyzed for progesterone to determine the interval to first ovulation and estrous cycle characteristics. Milk yield and concentrations of fat, protein, and lactose were measured every 2 wk. Cows were inseminated following visual observation of estrus. The breeding season commenced on April 8, 2009 and continued for 16 wk. Transrectal ultrasonography was carried out at 30 to 36 d and 60 to 66 d post-AI to diagnose pregnancy. The LE-CLA treatment resulted in a decrease in milk fat concentration (36.9±0.06 g/kg vs. 30.7±0.06 g/kg for control and LE-CLA, respectively) and yield (0.91±0.02 kg/d vs. 0.84±0.02 kg/d for control and LE-CLA, respectively); however, milk yield was increased by LE-CLA supplementation (24.7±0.7 kg/d vs. 27.2±0.7 kg/d for control and LE-CLA, respectively), resulting in no overall difference in milk energy output. No effect of LE-CLA was observed on any estrous cycle characteristics or measures of reproductive performance. These results support that in pasture-based systems of dairy production, where energy intake limits milk production, energy spared by CLA-induced milk fat depression is partitioned toward increasing milk yield rather than toward body reserves.

Effect of supplementation with different fat sources on the mechanisms involved in reproductive performance in lactating dairy cattle.

Supplementary fat positively influences reproductive performance in dairy cattle, although the mechanisms involved are not clearly defined. Our objective was to determine the effects of four different fat supplements on follicle development, plasma steroid hormone concentrations and prostaglandin (PG) synthesis in lactating dairy cattle. Forty-eight early lactation Holstein-Friesian cows (21 primiparous, 27 multiparous) were used in a completely randomized block design. Cows were fed the same basal TMR diet and received one of four fat supplements: (i) palmitic acid (18:0 fatty acid; Control), (ii) flaxseed (rich in 18:3 n-3 fatty acid; Flax), (iii) conjugated linoleic acid (a mixture of cis-9, trans-11 and trans-10, cis-12 isomers; CLA), and (iv) fish oil (rich in 20:5 and 22:6 n-3 fatty acids; FO). All lipid supplements were formulated to be isolipidic; palmitic acid was added as necessary to provide a total lipid supplement intake of 500 g/day. Cows were synchronized to be in estrus on Day 15 of dietary treatment. All antral follicles were counted, and dominant follicles, subordinate follicles and corpora lutea were measured daily via transrectal ovarian ultrasonography for one complete estrous cycle. Blood samples were collected daily, and selected samples were analyzed for progesterone, estradiol, insulin-like growth factor-1, insulin, cholesterol and non-esterified fatty acids. Estrus was synchronized a second time, and liver and endometrial biopsies were collected on Day 7 of the estrous cycle. Gene expression was evaluated for a number of genes involved in prostaglandin synthesis (endometrium) and fatty acid uptake and utilization (liver). Fat supplementation had little effect on follicle development. Cows receiving supplementary n-3 fatty acids had lesser plasma progesterone (P4) and smaller corpora lutea than cows receiving the CLA or Control supplements. Effects of fat supplementation on the endometrial expression of genes involved in PG synthesis were minor. Hepatic expression of SREBF1, ASCL1 and FABP1 was reduced by FO supplementation. Reduced plasma P4 in n-3 supplemented cows may lead to a suboptimal uterine environment for embryo development and hence reduced fertility compared to cows receiving the control or CLA supplements.

The effect of dry period duration and dietary energy density on milk production, bioenergetic status, and postpartum ovarian function in Holstein-Friesian dairy cows.

Following parturition, it is typical for dairy cows to enter a period of negative energy balance and body condition loss to support mammary milk synthesis; this is associated with compromised reproductive performance. Alternative management strategies during the prepartum (dry) and early postpartum periods may ameliorate this loss. Forty mature Holstein-Friesian cows were assigned to 1 of 2 dry period treatments [standard 8-wk dry period (SDP) or no planned dry period (NDP)] and 1 of 2 dietary energy density treatments [standard TMR (STMR) or high-quality TMR (HTMR)]. Milk yield during wk 1 to 12 postpartum was reduced in cows assigned to the NDP treatment. Energy balance and body condition score (BCS) during wk 1 to 4 postpartum were increased in cows assigned to the NDP treatment compared with the cows assigned to the SDP treatment, and BCS increased from wk 5 to 12 postpartum in the NDP cows compared with the SDP cows. During the first 12 wk postpartum, cows assigned to the HTMR diet had greater milk yield and reduced milk fat concentration compared with the cows assigned the STMR diet. The BCS was greater from wk 5 to 12 postpartum in HTMR cows compared with STMR cows. During the period from wk -3 to +3 relative to parturition, circulating concentrations of insulin, glucose, and IGF-I were greater in cows in the NDP treatment compared with cows in the SDP treatment. Cows assigned to the HTMR diet had greater circulating insulin and glucose concentrations compared with the STMR cows from wk -3 to +3 relative to parturition. The first postpartum ovulation occurred earlier for cows in the NDP treatment compared with cows in the SDP treatment (16.9 vs. 24.8 d postpartum. Cows assigned to the STMR diet tended to have a higher conception rate to first service compared with cows assigned to the HTMR diet. Energy balance and metabolic status can be improved by either eliminating the dry period or by feeding a higher energy diet, but effects on the reproductive axis appear to be different.

Embryo yield and quality following dietary supplementation of beef heifers with n-3 polyunsaturated fatty acids (PUFA).

The objective of this study was to examine the effects of dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on embryo yield and quality in heifers. Animals were individually offered barley straw and concentrate diets supplemented with either palmitic acid (C16:0; CON) or a partially rumen protected n-3 PUFA-enriched supplement. Following oestrous cycle synchronisation, superovulation was induced using FSH. Blood samples were collected for the measurement of fatty acids, metabolites, insulin and IGF-1. On day 7 post-insemination the number of ovulations was estimated and embryos recovered non-surgically and quality graded. At embryo recovery 50 ml of the uterine flushing was collected from each horn for fatty acid analysis. Grade 1 embryos were isolated, snap frozen in liquid nitrogen and stored at -80 degrees C. mRNA expression for six genes, LIF, BAX, Cx43 and E-CAD associated with embryo development, and PPAR-alpha and -delta, associated with lipid metabolism was analysed. The n-3 PUFA supplementation increased plasma n-3 PUFA concentration (P<0.05) and reduced n-6:n-3 PUFA ratio (P<0.05). Uterine concentration of the n-3 PUFA, eicosapentaenoic acid was increased (P<0.05) and the concentration of arachidonic acid decreased (P<0.05) following n-3 PUFA supplementation. While CON increased triglyceride concentrations, diet did not affect the other plasma metabolites, insulin or IGF-1 (P>0.05). Similarly, there was no effect of diet on superovulation rate, embryo recovery rate, embryo quality (P>0.05) or mRNA expression of the genes examined (P>0.05).

Development and quality of sheep embryos cultured in commercial G1.3/G2.3 sequential media.

Present study assesses the developmental ability and quality of ovine IVP embryos derived from culture in sequential media G1.3/G2.3. A total of 1474 cumulus-oocyte complexes were matured in M199 supplemented with EGF and FCS for 24h in 5% CO2 in humidified air at 39 degrees C. Oocytes were co-incubated in SOF medium with 1 x 10(6) spermatozoa/ml at the same temperature and gas conditions (Day 0 p.i.). Presumptive zygotes at 20 h p.i. were denuded, washed and placed in culture in SOF (control; n=742) or G1.3 media supplemented with 3mg/ml of BSA (n=732) under mineral oil in a humidified and controlled atmosphere at 39 degrees C. Embryos in the treated group were changed to G2.3 medium on Day 3 of culture. A group of blastocysts in each group were frozen by conventional method (SOF, n=55; G1.3/G2.3, n=48). In vivo embryos (n=72) were recovered at Day 7 from the uterus of progestagen+eCG treated females and they were cultured in defined medium (n=38) or frozen (n=34) directly after recovery. Cleavage rate of IVP embryos recorded at 48 h p.i. was similar for control and treated embryos (49.8 versus 47.5%). There were no significant differences in blastocyst development from the two groups on Day 6 (26.0 versus 25.6%), 7 (42.1 versus 38.6%) or 8 (50.8 versus 43.2%). Blastocyst development rates from total oocytes cultured were comparable (24.1 versus 21.5%). However, the proportion of hatched blastocysts was significantly higher for control embryos (86.6 versus 44.3%, P<0.0001). In addition, embryos cultured in SOF had higher re-expansion rates post-thawing at 24h (38.2 versus 6.2%), 48 h (36.4 versus 4.1%) and 72 h (34.5 versus 4.1%) and hatching rate (32.8 versus 2.0%) than embryos cultured in sequential media (P<0.0001). In vivo embryos showed higher hatching rate (61.7%) than IVP groups (SOF, P<0.01; G1.3/G2.3, P<0.0001) but lower than their fresh cultured counterparts (86.8%; P=0.01). In conclusion, G1.3/G2.3 media supported high developmental rates of embryos in vitro but the quality of the embryos was impaired.

Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression.

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-tau), and differentiation and implantation (LIF and LR-beta). 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-beta. 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-tau 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.

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.

Effect of diet quantity and urea supplementation on oocyte and embryo quality in sheep.

The objective was to investigate the effects of dietary energy and urea supplementation on oocyte and embryo quality in sheep using in vivo and in vitro experimental models. Sixty-three ewes were fed grass meal at 0.5 or 2.0 times maintenance energy requirements (MER). The diet was supplemented with feed grade urea (U) for half of the ewes on each energy treatment. Ewes were stimulated with 1000 IU eCG and either slaughtered on the day of pessary withdrawal, for in vitro embryo production, or mated and slaughtered on Day 5 for embryo recovery. Urea decreased cleavage rate (48.3 vs 39.7%) and consequently blastocyst rate (41.6 vs 36.8%) but the differences were not significant. Oocytes from animals on 2.0 MER had a lower cleavage rate (54.9 vs 36.0%) and blastocyst yield (49.3 vs 31.4%) than those on 0.5 MER. However, there was an interaction between urea and energy for cleavage (P = 0.04) and blastocyst yield (P = 0.03) indicating a variable response to urea in the presence of high energy. This was manifested by a decrease in cleavage rate in the presence of urea and high energy (22%, 8 of 36), and a reduction in blastocyst development (19%, 7 of 36). When blastocyst development rate was expressed as a proportion of cleaved oocytes there was no difference between groups; in addition, there was no difference between groups in terms of blastocyst hatching rate (overall mean 66.1%) or blastocyst cell number on Day 8 (overall mean +/- SEM, 138.4 +/- 9.0, n=61). The effect of urea on cleavage rate in vivo was more severe. Urea supplementation reduced (P<0.001) the cleavage rate (93 vs 62%). Despite this, the yield of blastocysts was unaffected. Oocytes from ewes on 0.5 MER exhibited a lower (P<0.05) cleavage rate than those on 2.0 MER (66 vs 87%). This effect was also apparent at the blastocyst stage (40.0 vs 50.9%), although the difference was no longer significant. There were no differences in hatching rate (overall mean 70.7%) or blastocyst cell numbers (overall mean +/- SEM, 166.3 +/- 15.6, n=40). Collectively, these results suggest that both high dietary energy and urea content influence subsequent embryo development in vitro, and the deleterious effects of urea are likely influenced by concomitant energy intake.

Effect of protein supplementation and presence of an antioxidant on the development of bovine zygotes in synthetic oviduct fluid medium under high or low oxygen tension.

The aim of this study was to investigate the effect of protein supplementation of culture medium and the presence of a putative antioxidant on bovine zygote development under 5% (low) and 20% (high) O2. In Experiment 1, presumptive zygotes (n=992) were cultured in synthetic oviduct fluid (SOF) alone or supplemented with 3 mg/mL PVP, 3 mg/mL BSA (SOFB), and/or 10% FCS (SOFBF) in 5% CO2, 5% O2, 90% N2. In Experiment 2, zygotes (n=1916) were cultured in SOF, SOFB or SOFBF with or without taurine under high and low O2. In Experiment 1, presence of BSA or BSA plus FCS significantly increased the speed of development compared to SOF or SOF+PVP. Blastocyst quality was also improved, as evidenced by increased hatching rate and cell numbers. In Experiments 2, taurine had no effect on development irrespective of oxygen concentration or protein supplementation. In conclusion, the presence of protein in the culture medium and culture under reduced O2 significantly improved embryo development. Taurine had no effect on development.

Bovine blastocyst production in vitro after inhibition of oocyte meiotic resumption for 24 h.

The aims of the present study were to assess the effect of various substances on meiotic resumption and subsequent development to the blastocyst stage of bovine oocytes. Immature cumulus-oocyte complexes were cultured for 24 h in (a) Medium 199 (M199) alone, or M199 supplemented with (b) 10% fetal calf serum (FCS), (c) 1 micrograms cycloheximide ml-1, (d) 2 mmol 6-dimethylaminopurine (6-DMAP) l-1, or (e) 0.1 mmol vanadate l-1. After 24 h, groups (a) and (b) were inseminated with frozen-thawed spermatozoa and subsequently cultured, while groups (c-e) were washed and cultured for a second 24 h in M199 + FCS, after which they were inseminated and cultured. At all time points a representative sample of oocytes were fixed and stained with orcein to observe the nuclear status, while others were labelled with [35S]methionine to study protein biosynthesis. Incubation with 6-DMAP, cycloheximide or vanadate completely blocked germinal vesicle breakdown with most oocytes remaining at the germinal vesicle stage after 24 h culture (89%, 100% and 85%, respectively). This inhibitory effect was fully reversible in the case of 6-DMAP and cycloheximide; after a second period of incubation, germinal vesicle breakdown occurred in almost all cases (99% and 100%, respectively), and most reached metaphase II (85% and 83%, respectively). In contrast, inhibition with vanadate was only reversible in 56% of oocytes, with only 6% reaching metaphase II. Cleavage rates at 72 h after insemination and blastocyst yields on day 8 of culture were, respectively: (i) M199, 72% and 34%; (ii) M199 + FCS, 80% and 45%; (iii) M199 + cycloheximide, 81% and 19%; (iv) M199 + 6-DMAP, 77% and 14%. 6-DMAP did not modify methionine incorporation. However, cycloheximide completely blocked protein synthesis when present during the period of labelling. Addition of epidermal growth factor to cycloheximide-inhibited oocytes was without effect. In contrast, epidermal growth factor overcame the effect of 6-DMAP in about 50% of oocytes, resulting in lower developmental rates after IVF. These results give an indication of the feasibility of in vitro meiotic inhibition as a tool in the study of the mechanisms involved in acquisition of competence.