Energy and lipid metabolism gene expression of D18 embryos in dairy cows is related to dam physiological status.

We analyzed the change in gene expression related to dam physiological status in day (D)18 embryos from growing heifers (GH), early lactating cows (ELC), and late lactating cows (LLC). Dam energy metabolism was characterized by measurement of circulating concentrations of insulin, glucose, IGF-1, nonesterified fatty acids, ß-hydroxybutyrate, and urea before embryo flush. The metabolic parameters were related to differential gene expression in the extraembryonic tissues by correlation analysis. Embryo development estimated by measuring the length of the conceptuses and the proportion of expected D18 gastrulating stages was not different between the three groups of females. However, embryo metabolism was greatly affected by dam physiological status when we compared GH with ELC and GH with LLC but to a lesser extent when ELC was compared with LLC. Genes involved in glucose, pyruvate, and acetate utilization were upregulated in GH vs. ELC conceptuses (e.g., SLC2A1, PC, ACSS2, ACSS3). This was also true for the pentose pathway ( PGD, TKT), which is involved in synthesis of ribose precursors of RNA and DNA. The pathways involved in lipid synthesis were also upregulated in GH vs. ELC. Despite similar morphological development, the molecular characteristics of the heifers' embryos were consistently different from those of the cows. Most of these differences were strongly related to metabolic/hormone patterns before insemination and during conceptus free-life. Many biosynthetic pathways appeared to be more active in heifer embryos than in cow embryos, and consequently they seemed to be healthier, and this may be more conducive to continue development.

Gene selection heuristic algorithm for nutrigenomics studies.

Large datasets from -omics studies need to be deeply investigated. The aim of this paper is to provide a new method (LEM method) for the search of transcriptome and metabolome connections. The heuristic algorithm here described extends the classical canonical correlation analysis (CCA) to a high number of variables (without regularization) and combines well-conditioning and fast-computing in "R." Reduced CCA models are summarized in PageRank matrices, the product of which gives a stochastic matrix that resumes the self-avoiding walk covered by the algorithm. Then, a homogeneous Markov process applied to this stochastic matrix converges the probabilities of interconnection between genes, providing a selection of disjointed subsets of genes. This is an alternative to regularized generalized CCA for the determination of blocks within the structure matrix. Each gene subset is thus linked to the whole metabolic or clinical dataset that represents the biological phenotype of interest. Moreover, this selection process reaches the aim of biologists who often need small sets of genes for further validation or extended phenotyping. The algorithm is shown to work efficiently on three published datasets, resulting in meaningfully broadened gene networks.

Expression of nuclear and membrane progesterone receptors in the canine oviduct during the periovulatory period.

Important reproductive events take place in the canine oviduct in the presence of increasing concentrations of progesterone (P4). To investigate the potential effects of P4 on the canine oviduct, the expression of nuclear (PR) and membrane (PGRMC1 and 2, mPRα, ß and γ) P4 receptors was studied by quantitative RT-PCR and immunohistochemistry. Oviducts were collected from Beagle bitches after the onset of pro-oestrus and before the LH peak (Pre-LH), after the LH peak and before ovulation (Pre-ov) and on Days 1, 4 and 7 post-ovulation (n=6 bitches/stage). PR mRNA concentrations decreased from Pre-LH to Day 7 in the ampulla and isthmus, whereas both PGRMC1 and 2 mRNA levels increased over the same period. The main change in mPR expression was an increase in mPRß and γ mRNAs at Day 7 in the isthmus. Furthermore, PR proteins were expressed in the nuclei of luminal epithelial, stromal and muscular cells, whereas the expression of PGRMCs and mPRs was primarily cytoplasmic and localised in the luminal epithelium. The immunostaining for PR decreased at Day 4 in the stroma and muscle, whereas it remained strong in the epithelium from Pre-LH to Day 7. PGRMC1 staining was strong at Days 4 and 7 whereas PGRMC2 was highly expressed from Pre-ov to Day 7. The most intense immunostaining signals for all three mPRs were observed at Day 7. Our results strongly support the hypothesis that P4 is an important regulator of oviductal functions in the bitch through complementary classical and non-classical P4 pathways.

Postpartum variations of plasma IGF and IGFBPs, oocyte production and quality in dairy cows: relationships with parity and subsequent fertility.

The aim of this study was to determine whether postpartum variations of plasma IGF-1 and IGFBP concentrations, oocyte production and quality were related to parity and subsequent conception rate in Holstein dairy cows. Holstein dairy cows [10 primiparous (PP) and 22 multiparous (MP)] were allotted in six batches and sampled once weekly between calving and oestrous synchronization treatment started at 71.2 ± 2.0 days postpartum. During the 3 weeks before treatment, ovum pick-up (OPU) was performed twice weekly. Oocytes were scored on a 4-point scale, and oocytes from OPU1, 3 and 5 were fertilized in vitro. Seventeen cows became pregnant after first and second AI and were considered as fertile (F), while the others were considered to be subfertile (SF). Logistic regression was carried out to investigate the relationships between repeated measurements and fertility including parity and batch effects in the models. Likelihood of fertility significantly increased when plasma urea and IGFBP-3 concentrations decreased and was higher in PP compared with MP cows. There was a trend for fertility to increase when plasma IGF-1 concentrations increased (p = 0.07). In vitro cleavage and development rates were similar between SF and F cows (46.4% and 28.3% in SF vs 55.0% and 22.1% in F). Parity had an effect on plasma IGF-1 concentrations (PP: 61.65 ± 2.67 vs MP: 41.63 ± 5.81 ng/ml, p < 0.001), mean number of follicles aspirated per session (PP: 5.7 ± 1.3 vs MP: 9.5 ± 0.8, p < 0.05) and fertility (PP: 8/10 = 80% vs MP: 9/22 = 41%, p < 0.05) but not on the number of oocytes recovered per session nor their quality. In conclusion, postpartum plasma urea and IGFBP-3 concentrations, but not oocyte production and quality before breeding, were related to subsequent conception rate in our experimental design. Parity had a significant effect on energy status, follicular growth and fertility and needs to be considered when investigating relationships between nutrition and reproduction.

Pre- and post-partum mild underfeeding influences gene expression in the reproductive tract of cyclic dairy cows.

Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post-partum. This study investigated the effects of pre- and post-partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post-partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post-partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi- and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post-partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post-partum patterns of IGF-1, insulin, ß-hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid-related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post-partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post-partum dairy cows.

Are oocytes from the anestrous bitch competent for meiosis?

In the bitch, oocyte meiosis resumption takes place in the oviduct. Using oocytes from anestrous bitches, in vitro maturation (IVM) generally gives very poor results. To investigate the contribution of oocyte competence to the low IVM yield, we compared in vivo maturation in an optimal environment with conventional IVM. A total of 418 grade 1 cumulus-oocyte complexes (COCs) from 10 anestrous bitches were transferred into the oviducts of recipient bitches either on Day -1 (n = 3 recipients), Day 0 (n = 2) or on Day +1 (n = 2) relative to ovulation. For each donor bitch, 20 grade 1 COCs were also cultured in vitro. After 72 h of in vivo or IVM, the nuclear stage of oocytes was determined after DNA and tubulin staining. Of the 154 oocytes recovered and examined after intratubal transfer, only 2% reached the metaphase I or II stage and 38.3% were degenerated. Oocytes cultured in vitro displayed a higher metaphase rate (7.6%, n = 170) and lower degeneration rate (12.9%) compared with transferred oocytes (p < 0.001). These results clearly demonstrate that the oocyte competence is the major limiting factor of IVM efficiency in the dog. Mimicking the tubal environment may thus not be sufficient to increase IVM yield in this species.

Kick-starting ovarian cyclicity by using dietary glucogenic precursors in post-partum dairy cows: a review.

The objective of this review is to describe how dietary glucogenic precursors could stimulate ovarian activity in post-partum dairy cows and improve reproductive success. Although the nutrient requirements for the early resumption of ovarian cycles, and for follicle and embryo development are quantitatively small, reproductive success is deteriorated by post-partum negative energy balance. Since very little glucose is absorbed directly from the digestive tract of ruminants one of the targets for nutritional manipulation could be the glucogenic potential of the diet. This could be achieved by giving rumen-resistant starch or mono-propylene glycol. Both these adaptations increase glucose, insulin and insulin-like growth factor-1 plasma concentrations and stimulate ovarian follicle growth.

Relationships between welfare and reproductive performance in French dairy herds.

This study aimed to investigate the relationship between cow reproductive performance and welfare evaluated at the herd level using the Welfare Quality protocol. The 11 criteria, four principles (good feeding, good housing, good health and appropriate behavior, scale 0-100) and overall welfare category (excellent/enhanced/acceptable/not classified = poor welfare) were included as risk factors for calving to first service interval (CFSI) and calving rate (CR). The confounding factors cow breed, parity, season of calving and AI, calving to AI interval, rank of AI (1-3) and milk production were taken into account. The sample included 3951 AIs (2172 AI1, 1182 AI2, 597 AI3) in 124 French commercial dairy herds. Median CFSI was shorter for the cows bred in herds with a higher overall welfare category (median 75 and 76 days in enhanced and acceptable herds vs. 86 in poor welfare ones, P = 0.02). The scores for absence of injuries and expression of social behavior tended to be associated with CFSI (P < 0.10). Calving rate (34.5%) was not related to the overall welfare category. However, CR was positively related to the good housing score and a positive trend was observed with the scores for absence of prolonged hunger and absence of injuries. In conclusion, this study confirms a positive relationship between CFSI and welfare in dairy cows with no explicit links with specific aspects of animal welfare.

Progesterone-releasing devices for cattle estrus induction and synchronization: Device optimization to anticipate shorter treatment durations and new device developments.

Synchronization programs using progesterone-releasing intravaginal devices that allow for fixed time artificial insemination are still finding increasing application in bovine reproduction. This practice is useful for rationalizing livestock management because an increased number of cows can be inseminated in one session without the need for estrus detection. Although much of the innovation related to the design and development of intravaginal devices for use in cattle took place in the previous century, progress in understanding the physiology of the bovine estrous cycle resulted in shorter treatment durations, a trend which is still continuing. In this competitive market, with little functional differentiation between the existing devices, the shorter treatment duration prompted for optimization of the progesterone content in the device, as the cost of the drug significantly contributes to the price per unit. For CIDR® a reduction of the progesterone content of about 30 per cent was realized. Price reduction remained an important target for further device development. Next to reduction of progesterone content, cheaper and easier to process materials like polyethylene vinyl acetate (EVA) copolymers have been explored to replace the commonly used silicone elastomers. The reengineering effort of CIDR® demonstrated that knowledge of release kinetics and insight into gradual depletion patterns in the device is critical for optimization of drug content without compromising performance (blood levels). More recent publications related to the use of alternative polymers like EVA and polyisoprene (IP) indicated encouraging results regarding further reduction of progesterone content. The use of EVA seems most promising, because it is in principle a low-cost polymer available in many grades and this thermoplastic polymer can be processed easily by means of commonly used techniques like injection molding and extrusion. The use of thermoplastic polymers, however, requires insight into the physical-chemical phenomena related to drug dissolution and re-crystallization taking place in the polymer during processing at high temperatures. These aspects, which may critically affect product stability, are often overlooked and are prompting to cover some of the background in this review. Finally, two different innovative approaches are discussed, one related to programable electronic devices for tailored simultaneous drug release and the other is a flexible drug-loaded helix, which is retained very well in several species without causing the usual inflammatory response.

Genetic and environmental factors influencing first service conception rate and late embryonic/foetal mortality in low fertility dairy herds.

The objective of this study was to identify factors affecting variation in conception rate to first artificial inseminations (AI) (CR: number of pregnant cows on D80-100/inseminated cows) and the incidence of embryonic/foetal loss (LEM) between 21 and 80 days of pregnancy (number of cows non-pregnant on D80-100/pregnant on D21) in 44 low fertility dairy herds of the west-central region of France. Reproductive status was assessed using progesterone milk concentration on D0 = Day of AI and D21-24, plasma PSPB concentration on D30-35, rectal palpation on D80-100 and observed return to oestrous. The final data set contained 1285 Prim'Holstein cows, 5.0% (64/1285) were inseminated in the luteal phase (progesterone > or = 3 ng/ml on D0), 61.3% (787/1285) were pregnant on D21-24 (progesterone < 3 ng/ml on D0 and > or = 5 ng/ml on D21-24), 15.4% lost their embryo/foetus between D21-24 and D80-100 (198/1285) and 45.8% (589/1285) were pregnant on D80-100. The incidence of late embryonic/foetal loss (LEM) was 25.2% (198/787). Multivariate logistic regression models including the random herd effect were used to analyse the relationship between AI centre, AI sire, cow's sire, parity, interval between calving and AI, milk production, milk protein content, body condition score (BCS) on D0, season of calving, season of AI, estimated genetic index on CR and LEM incidence. CR was significantly related to parity (p < 0.05), milk production after calving (p < 0.05) and estimated genetic value (p < 0.01). A significant difference in CR was observed for calving to AI interval > or = 70 days versus > or = 90 days, but the overall effect of the interval was not significant (p = 0.11). LEM incidence was affected by period of AI (p < 0.05), milk production (p < 0.05) and BCS (p < 0.05), but was not related to estimated genetic index. In conclusion, in these low fertility herds, the incidence of LEM was high and 25% of the cows lost their embryo after 21 days of pregnancy. LEM was affected by specific factors (season, BCS), which were not related to CR. The absence of a relationship between estimated genetic index and LEM in spite of its effect on CR indicates that estimated genetic merit has a greater effect on early embryonic loss or fertilisation failure than on later stages of embryo development.

Interferon stimulated genes as peripheral diagnostic markers of early pregnancy in sheep: a critical assessment.

We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0=day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 (CXCL10), Myxovirus (influenza virus) resistance 1 (MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher (P0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 (MX2) and ISG15 ubiquitin-like modifier (ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene® tubes on farms, for early pregnancy detection in sheep.

Sire effect on early and late embryonic death in French Holstein cattle.

We investigated the effect of maternal sire on early pregnancy failure (between D0, day of insemination and D90) in their progeny during the first and second lactations (n=3508) in the Holstein breed. The estimated breeding value (EBV) for cow fertility of 12 bulls (reliability⩾0.95) was used to create the following three groups: low, medium and high EBV (EBV from -0.7 to 1 expressed as genetic standard deviation relative to the mean of the breed). In their daughters (93 to 516 per bull), progesterone measurement was carried out on the day of artificial insemination (AI; D0) to check whether the cows were in the follicular phase and on D18 to 25 to assess non-fertilisation-early embryonic mortality (NF-EEM). Late embryonic mortality (LEM) and early foetal death (FD) were determined by ultrasonography on D45 and D90 and by the return to oestrus after the first AI. Frequencies of NF-EEM, LEM, FD and pregnancy were 33.3%, 11.7%, 1.4% and 48.5% and incidences were 35.1, 19.0, 2.7 and 51.1, respectively. Sire EBV was significantly related to the incidences of pregnancy failure between D0 and D90, fertilisation failure-early embryonic mortality (FF-EEM) and LEM but not to the incidence of FD between D45 and D90 of pregnancy. The relative risk (RR) of FF-EEM was significantly higher (RR=1.2; P<0.05) for the progeny group of low EBV bulls when compared with high EBV bulls. The same effect was observed when comparing LEM of the progeny groups from the low EBV bulls to those from moderate and high EBV bulls (RR, respectively, of 1.3 and 1.4; P<005). The incidence of FF-EEM was significantly higher when cows were inseminated before 80 days postpartum compared with later, and for the extreme values of the difference between milk fat and protein content measured during the first 3 months of lactation. FF-EEM was also significantly related to the year of observation. The incidence of LEM was higher for the highest producing cows and was influenced by interaction between milk yield×lactation rank and milk yield×milk protein content. In conclusion, this study showed large differences in early pregnancy failure between progeny groups and highlights the interest of accurate characterisation of embryonic death in order to identify potential candidate genes for female fertility.

Expression of components of the insulin-like growth factor system and gonadotropin receptors in bovine cumulus-oocyte complexes during oocyte maturation.

IGF system expression has been largely explored in the bovine follicular wall whereas it remains poorly studied in the COC. Using semi-quantitative RT-PCR and Western blot analysis, we have investigated spatial and temporal expression of IGF-1, IGFR-1, IGFBP-2, IGFBP-4, as well as gonadotropin receptors in bovine COC during oocyte maturation. In addition, we have compared changes in the IGF system and FSHR expression during in vitro maturation in TCM199 alone or in the presence of 10 ng/ml of EGF. The transcripts for IGFR-1 and IGFBP-2 were detected in cumulus and germinal cells whereas IGF-1, IGFBP-4 and FSHR mRNA were restricted to cumulus cells. Topography of the IGF system and gonadotropin receptor expression within COC were unaffected by the maturation step. In contrast, levels of IGFBP-2 and FSHR expression decreased (P < 0.05) in matured COC. Under defined culture conditions, IGFBP-2 and FSHR mRNA expression remained at a high level in TCM199 alone and were significantly reduced (P < 0.05) in the presence of 10 ng/ml EGF after a 24 h period of in vitro maturation. In conclusion, our results demonstrate a cell-specific pattern of IGF system member gene expression within bovine COC suggesting interaction between the somatic and germinal compartments. In addition, synchronized changes in the pattern of COC IGFBP-2 and FSHR expression during oocyte maturation suggest possible synergistic actions between IGF-1 and FSH.

Influence of the type of energy supply on lh secretion, follicular growth and response to estrus synchronization treatment in feed-restricted suckler beef cows.

The present experiment aimed to compare the efficiency of supplementation (+17.5 MJ Net Energy/d starting 47 +/- 4 days after calving) with concentrate (CS, maize grain, n = 10) or with forage (FS, maize silage, n = 10) in estrus-synchronized (Norgestomet implant 10 days inserted 60 +/- 4 days postpartum + PMSG at implant removal) beef cows previously restricted (47 MJ Net Energy/d, 785 g CP/d, 70% of requirements). The type of diet had no significant effect on basal LH concentrations (CS: 0.18 +/- 0.12 vs FS: 0.11+/- 0.02 ng/mL), LH pulse frequency (CS : 0.7 +/- 0.3 vs FS: 0.8 +/- 0.2 pulse/10 h), LH pulse amplitude (CS: 0.55 +/- 0.50 vs FS : 0.62 +/- 0.50 ng/mL) or estradiol (E2) concentrations (CS: 3.3 +/- 0.8 vs FS: 4.6+ /- 0.8 pg/mL) 13 days after the beginning of energy supplementation. No differences between CS and FS cows were observed for the number of small, medium and large follicles nor on the size of the largest follicle from 11 days before implant insertion to implant removal (IR). After IR, an LH surge was observed in 2 of the CS and 4 of the FS cows. The type of energy supplementation had no significant effect on LH (CS: 0.16 +/- 0.06 ng/mL vs FS 0.48 +/- 0.06 ng/mL; P > 0.05) or on estradiol concentrations (CS : 7.8 +/- 0.2 vs FS : 8.9 +/- 0.2 pg/mL, P > 0.10) measured hourly from 29 to 49 h after IR. Cows that ovulated after IR tended to have higher E2 concentrations than cows that did not ovulate (9.4 +/- 0.2 vs 6.3 +/- 0.2 pg/mL, P = 0.08). Similar ovulation and pregnancy rates were observed in CS and FS cows (CS: 6/10 vs FS: 7/10 and CS: 6/10 vs FS: 5/10 respectively, P > 0.05). To conclude, energy supplementation with forage was as effective as energy supplementation with concentrate to influence follicular growth, ovulation and pregnancy percentage after estrus synchronization treatment in diet-restricted beef cows.

Estrus synchronization in beef cows: comparison between GnRH+PGF2alpha+GnRH and PRID+PGF2alpha+eCG.

The aim of this study was to compare two protocols for estrus synchronization in suckled beef cows over a 2 years period. The population studied consisted of 172 Charolais and 168 Limousin cows from 12 and 14 beef herds, respectively. In each herd, cows were allotted to groups according to parity, body condition score and calving difficulty. Cows in Group 1 (n=174) received PRID on Day-8 with estradiol benzoate (10mg, vaginal capsule), dinoprost on Day-4 (25mg i.m.), eCG on Day 2 (500 IU i.m.). The PRID was removed on Day-2 and cows were inseminated on Day 0, 56 h after PRID was removed. Cows in Group 2 (n=166) received GnRH on Day-10 (100 microg i.m.), dinoprost on Day-3 (25mg i.m.) and GnRH on Day-1 (100 microg i.m.), and were inseminated on Day 0, 16-24h after the last GnRH treatment. Plasma progesterone concentrations were measured to determine cyclicity prior to treatment (Days-20 and -10), to confirm the occurrence of ovulation (Days 0 and 10) and to determine the apparent early pregnancy rate (Days 0, 10 and 24). Pregnancy diagnosis was performed by ultrasonography between Days 35 and 45. The effects of various factors on ovulation, apparent early pregnancy and pregnancy rates were studied using logistic mixed models. There was no significant difference between Groups 1 and 2, respectively, for the cyclicity rate before treatment (80.5% versus 80.1%), for apparent pregnancy rate on Day 24 (62.1% versus 54.8%, P=0.09) and for pregnancy rate on Days 35-45 (53.8% versus 46.3%, P=0.16). Ovulation rate was higher (P<0.01) in Group 1 (90.8%) than in Group 2 (77.1%) and was affected by cyclicity prior to treatment in Group 2 but not in Group 1 (Group 1: 88.2% in anestrous cows versus 91.4% in cyclic cows; Group 2: 45.5% in anestrous cows versus 85.0% in cyclic cows, P interaction=0.05). Apparent pregnancy rates on Day 24 were influenced by the year of study (52.4% versus 68.8%, OR=2.12, P<0.01) and by the cyclicity before treatment (anestrous cows 46.3% versus cyclic cows 61.5%, OR=1.86, P<0.05). Pregnancy rates at 35-45 days were influenced by the year of study (44.2% versus 59.8%, OR=1.92, P<0.01). In conclusion, although pregnancy rates were similar for the two treatments, the combination of GnRH+PGF2alpha+GnRH in suckled beef cows induced a lower rate of ovulation than treatment with PRID+PGF2alpha, particularly in anestrous cows.

Sources of variation of post-partum cyclicity, ovulation and pregnancy rates in primiparous Charolais cows treated with norgestomet implants and PMSG.

In this study, sources of variation of postpartum cyclicity, ovulation and pregnancy rates were analyzed for 723 primiparous suckled Charolais cows treated with combined norgestomet implants (Crestar) and 600 IU PMSG (Chronogest) injected at the time of implant removal. The cows were inseminated 48 and 72 h after implant removal. Cyclicity and ovulation rate were estimated by progesterone assay and pregnancy rate by ultrasonography. At time of implant insertion, difficulty of previous calving, body condition score (BCS, from 1 to 5), interval from calving to implant insertion and herd related factors were recorded and their effects analyzed by logistic regression models. Cyclicity, ovulation and pregnancy rates were, respectively, 14.7% (106/723 ), 67.1% (381/568 ) and 42% (303/722 ) and were affected by BCS, calving conditions and interval from calving to implant insertion (P values from < 0.01 to < 0.0001). For ovulation and pregnancy rate, an interaction between BCS and interval from calving to implant insertion was found (P < 0.01). No other main factor or interaction was found to be significant. Cyclicity rate was lower in BCS1 (score < 2.5) cows (9.6%) than in BCS2 (19.8%) or BCS3 (score > 2.5) cows (22.4%), and decreased as difficulty of calving increased (23.2, 13.6 and 10.1%, respectively, for calving conditions 1, 2 and 3 cows). Cyclicity rate increased with interval from calving to implant insertion (8.2, 10.2 and 19.5%, respectively, for interval from calving to implant insertion < 60 d, between 60 and 70 d and > 70 d). Similar trends were found for ovulation rate. Previous difficult calving conditions influenced pregnancy rate negatively (47.9, 43.8 and 32.5% for calving conditions 1, 2 and 3 cows, respectively; P < 0,005).

Influence of flushing on LH secretion, follicular growth and the response to estrus synchronization treatment in suckled beef cows.

The effects of energy supplementation (flushing) on LH and estradiol secretion, follicular growth and the response to estrus synchronization treatment (Norgestomet + PMSG initiated 41.9 +/- 3.4 d after calving) were investigated in 16 suckled beef cows fed either 70% (Group C, n = 8) of energy requirements from calving to 3 wk after AI or fed the same restricted diet until 11 d before synchronization and then were supplemented with 2 kg concentrate until 3 wk after AI (Group S, n = 8). Concentrations of LH and estradiol 17 beta were measured from 3 sampling periods: 25 and 39 d after calving and between 29 and 49 h after implant removal. Ovaries were examined by ultrasonography 11 d before treatment to implant withdrawal (IR). The effects of energy level, day (or hour) of observation and corresponding interactions were tested on repeated measurements by split-plot ANOVA. No positive effect of flushing was observed on characteristics of LH secretion on Day 39. However, the size of the largest follicle and the number of large follicles were higher in Group S than in Group C cows, respectively, 7 and 9 d after the beginning of flushing to 2 d after the start of treatment. After IR, the estradiol secretion tended to be higher in Group S than in Group C cows (9.8 +/- 0.4 pg/mL vs 7.2 +/- 0.2 pg/mL; P = 0.06), but no effect on LH secretion was observed. After implant removal 12 cows ovulated (Group S: 7/8 vs Group C: 5/8; P > 0.05), 7 were pregnant at 21 d after AI (Group S: 6/8 vs Group C: 1/8; P < 0.05) and 4 at 45 d after AI (Group S: 4/8 vs Group C 0/8; P > 0.05). To conclude, flushing had a positive effect on follicular growth, which does not seem to be mediated by LH. In cows fed a restricted diet, flushing enhanced follicular growth, increased the fertilization rate and/or reduced early embryonic death.

Effect of blood handling conditions on progesterone assay results obtained by chemiluminescence in the bitch.

Assay of blood progesterone (P4) is commonly practiced to determine the time of ovulation, diagnose luteal insufficiency, and predict time of parturition in bitches. Because of practical constraints, most blood samples cannot be assayed on site immediately after collection. The aim of this work was to evaluate the effects of various sampling and storage conditions on concentrations of P4 as determined by chemiluminescence immunoassay. The blood of 5 Beagle bitches was collected from the jugular vein to study the effect of the type of collection tube (silicone, lithium heparin, EDTA), the storage time of unseparated or separated plasma (2 h to 14 d), and the number of freeze-thaw cycles (1-10) on P4. The effect of each factor was tested within one assay session. None of the factors significantly affected P4. Thus, P4 appears to remain relatively stable in canine blood samples exposed to various processing and storage conditions.

Oocyte and embryo production and quality after OPU-IVF in dairy heifers given diets varying in their n-6/n-3 fatty acid ratio.

Dietary fat supplementation can improve oocyte quality in ruminants. The influence of the type of dietary fat on the number and quality of oocytes collected by ovum pick-up and on the production of embryos in vitro was investigated in Holstein heifers. Heifers were given hay plus one of two dietary supplements for 42 days. The supplements were linseed (L, rich in linolenic acid, C18:3n-3, n = 9) or soya bean (S, rich in linoleic acid, C18:2n-6, n = 9). Oocytes were collected by ovum pick-up (OPU) for 6 wks (2 sessions/wk) and morphologic quality assessed. Half the oocytes were frozen and the other half was used to produce embryos. Blood samples were analyzed for: insulin, insulin-like growth factor-1, glucose, non-esterified fatty acids, ß-hydroxy butyrate and urea and the proportions of fatty acids. Neither growth rate nor plasma hormone and metabolite concentrations were affected by dietary supplement. However, L significantly increased the proportion of plasma C18:3n-3 while S significantly increased the proportion of C18:2n-6(P < 0.001). Neither oocyte characteristics (number, their quality and number fertilized and cleaved per heifer per session) nor embryo characteristics (number and quality per heifer per session) and embryo development stages were affected by dietary treatment. Real-time RT-PCR was performed on immature and mature cumulus-oocyte complexes (COC). Prostaglandin E synthase-1 expression increased in L compared to S heifers. In conclusion, the type of fatty acid did not modify the numbers of oocytes and embryos produced by OPU-IVF and their quality in dairy heifers. Upregulation of prostaglandin E synthase-1 may ensure sufficient PGE(2) production for oocyte maturation even when its precursor is low.

Reduction of body-weight gain enhances in vitro embryo production in overfed superovulated dairy heifers.

The aim of our study was to test whether a reduction in dietary intake could improve in vitro embryo production in superovulated overfed dairy heifers. Cumulus-oocyte complexes of 16 Prim' Holstein heifers (14 +/- 1 months old) were collected by ovum pick-up (OPU), every 2 weeks following superovulation treatment with 250 microg FSH, before being matured and fertilized in vitro. Embryos were cultured in Synthetic Oviduct Fluid medium for 7 days. Heifers were fed with hay, soybean meal, barley, minerals and vitamins. From OPU 1 to 4 (period 1), all heifers received individually for 8 weeks a diet formulated for a 1000 g/day live-weight gain. From OPU 5 to 8 (period 2), the heifers were allocated to one of two diets (1000 or 600 g/day) for 8 weeks. Heifers' growth rates were monitored and plasma concentrations of metabolites, metabolic and reproductive hormones were measured each week. Mean live-weight gain observed during period 1 was 950 +/- 80 g/day (n = 16). In period 2 it was 730 +/- 70 (n = 8) and 1300 +/- 70 g/day (n = 8) for restricted and overfed groups respectively. When comparing period 1 and period 2 within groups, significant differences were found. In the restricted group, a higher blastocyst rate, greater proportions of grade 1-3 and grade 1 embryos, associated with higher estradiol at OPU and lower glucose and beta-hydroxybutyrate, were observed in period 2 compared with period 1. Moreover, after 6 weeks of dietary restriction (OPU 7), numbers of day 7 total embryos, blastocysts and grade 1-3 embryos had significantly increased. On the contrary, in the overfed group, we observed more <8 mm follicles 2 days before superovulation treatment, higher insulin and IGF-I and lower nonesterified fatty acids in period 2 compared with period 1 (no significant difference between periods for embryo production). After 6 weeks of 1300 g/day live-weight gain (OPU 7), embryo production began to decrease. Whatever the group, oocyte collection did not differ between period 1 and 2. These data suggest that following a period of overfeeding, a short-term dietary intake restriction (6 weeks in our study) may improve blastocyst production and embryo quality when they are low. However, nutritional recommendations aiming to optimize both follicular growth and embryonic development may be different.