G.C. van der Weijden

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory lh surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.

Insulin-like growth factor-I (IGF-I) stimulates the development of cultured rat pre-antral follicles.

The aim of the present study is to investigate the effect of insulin‐like growth factor (IGF‐I) on the development of cultured rat pre‐antral follicles. Pre‐antral follicles with a diameter between 140 and 160 μm are mechanically isolated from ovaries of 10‐day‐old rats and cultured in groups for 6 days in FSH and insulin‐containing serum‐free medium in the absence or presence of IGF‐I at concentrations of 1, 10, and 100 ng/ml, respectively. DNA content of the follicles before and after culture is measured to evaluate whether a possible growth is due to proliferation of follicular cells and the ultrastructure of the cultured follicles is studied with transmission electron microscopy to evaluate the quality of follicles cultured under different conditions. Furthermore, reverse transcriptase polymerase chain reaction (RT‐PCR) is used to assess the gene expression of IGF‐I and type I IGF receptor in pre‐antral follicles. When follicles were cultured in medium containing IGF‐I at a concentration of 1 and 10 ng/ml, the increase in follicle diameter after a 6‐day culture differs significantly from that in the absence of IGF‐I. The ultrastructure of follicles cultured in IGF‐I‐containing medium is better sustained when compared to that of follicles cultured in the absence of IGF‐I. Especially, theca cells, which are undergoing degeneration under controlled culture condition, demonstrate normal cellular ultrastructure with some characteristics of steroid‐secreting cells in the presence of IGF‐I. Moreover, in oocytes of follicles cultured in the presence of IGF‐I, cortical granules are observed distributed along the ooplasma membrane whereas cortical granules are hardly present in follicles cultured without IGF‐I. With RT‐PCR, the presence of the mRNA of IGF‐I and type I IGF receptor is demonstrated in both the somatic follicular cells and the oocytes. Taken together, these results suggest that IGF‐I, in the presence of follicle‐stimulating hormone (FSH), enhances rat pre‐antral follicle development in vitro and supports the morphology of cultured pre‐antral follicles. The stimulatory effect of exogenous IGF‐I on the development of pre‐antral follicle together with the gene expression of both IGF‐I and type I IGF receptor in pre‐antral follicles suggests the involvement of IGF‐I in early folliculogenesis and its actions are likely mediated by the putative type I IGF receptor, via both endocrine and paracrine/autocrine pathways. Mol. Reprod. Dev. 58:287–296, 2001.

Immunohistochemical localisation of growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I) and type I IGF-I receptor, and gene expression of GH and GHR in rat pre-antral follicles.

We previously demonstrated that the development of cultured rat pre-antral follicles is stimulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I) and that the mRNA of IGF-I and type I IGF receptor (IGFR) is present in the oocyte and wall of these follicles. To gain a closer insight into the regulation of early folliculogenesis by GH and IGF-I, the present study investigated the gene expression of GH and GHR mRNA in isolated oocytes and follicular wall cells of pre-antral follicles, using reverse transcriptase polymerase chain reaction, and the localisation of immunoreactive IGF-I, IGFR, GH and GHR proteins in ovarian sections of 10-day-old rats. GH was detected in oocytes and follicular wall tissue of pre-antral follicles, whereas expression of the GH mRNA was absent. The GHR mRNA was present in follicular wall tissue and not in the oocyte, while positive immunostaining for GHR was observed in all cells of the pre-antral follicles. Immunoreactive IGF-I and IGFR was also visible in the pre-antral follicles, especially in the oocytes. In conclusion, the data show that the previously demonstrated local gene expression of IGF-I and IGFR in oocytes and their enveloping follicular cells also leads to translation, which points to the involvement of intrafollicular IGF-I in early follicular development. The presence of the GHR mRNA and the GHR and GH proteins in pre-antral follicles in the absence of ovarian GH mRNA suggest a direct effect of systemic GH on early follicular development.

In vitro culture of rat pre-antral follicles with emphasis on follicular interactions

The present study was designed to determine whether rat pre‐antral follicles can grow under in‐vitro conditions. Emphasis is on whether follicular interaction is involved in in‐vitro follicle culture, and furthermore its role in follicular development has been assessed. Pre‐antral follicles were isolated mechanically from 10‐day old rat ovaries. They were divided into small (50 μm < diameter < 100 μm) and large (120 μm < diameter < 200 μm) pre‐antral follicles and cultured individually or in groups for 6 days in medium with or without fetal calf serum (FCS). Based on morphological criteria, large pre‐antral follicles cultured in groups in serum‐free medium had significantly higher survival rates than those cultured individually. In the presence of FCS, no significant difference was detected with respect to the survival. However, the large pre‐antral follicles cultured in groups had a significantly greater increase in diameter than those cultured individually. Furthermore, follicles cultured in groups in FCS‐containing medium exhibited significantly more follicular cell proliferation than those in serum‐free medium, based on DNA measurement. The present culture system (with or without FCS) proved to be insufficient for small pre‐antral follicles to stimulate growth comparable to that of large pre‐antral follicles. The transmission electron microscopical (TEM) study revealed the ultrastructural differences between follicles cultured in FCS‐containing and serum‐free media. Taken together, the results suggest that interfollicular factors are involved in follicle development in vitro, which especially at the early folliculogenesis stage plays a positive role in terms of follicular growth as well as survival. The present culture model allows further investigation of factors that regulate early folliculogenesis. Mol. Reprod. Dev. 55:65–74, 2000.

Changes in Water Content, Collagen Degradation, Collagen Content, and Concentration in Repeated Biopsies of the Cervix of Pregnant Cows

Abstract The objective of the present study was to assess if cervical ripeness could be quantified by measuring the percentage of denaturation of the collagen network of the stromal layer. Biopsy specimens from the caudal part of the cervix were obtained from nine pluriparous cows between Days 149 and 157 of gestation (second-trimester biopsy), at exactly Day 275 of gestation (term biopsy), and shortly after calving (calving biopsy). The samples were divided into a superficial stromal part and a deep stromal part. The water content was derived from the weight of the samples before and after lyophilization. A colorimetric assay was used to assess the percentage of collagen denaturation by determining the extinction at 570 nm of hydroxyproline released from α-chymotrypsine-treated samples. By incorporating a hydroxyproline standard series in the measurements, the insoluble collagen content (µg/mg dry wt) as well as the insoluble collagen concentration (µg/mg wet wt) could be derived. The water content of both layers of the cervix significantly increased between midpregnancy and parturition (P < 0.01). The insoluble collagen content and the insoluble collagen concentration were significantly increased at term (P < 0.01 and P < 0.05, respectively) but were significantly decreased at calving (P < 0.05 and P < 0.01, respectively). Both parameters showed no significant differences between the superficial and deep stromal layer, and they were significantly correlated with each other. A significant increase in the percentage denaturation of the deep stromal layer occurred between the second trimester and term pregnancy (P < 0.01), whereas at calving, the percentage denaturation had not significantly increased compared to term. The percentage of collagen denaturation of the superficial stromal layer did not significantly change with stage of gestation or at parturition. Our findings indicate that cervical ripening is a combination of increased collagen synthesis and increased percentage of collagen denaturation, whereas at calving, an increased digestion of the denatured collagen leads to increased collagen loss from the cervical connective tissue. The finding that cervical ripening mainly takes place in the deep stromal layer of the cervix emphasizes the importance of a detailed description of the tissue sampling sites for a proper interpretation of the results obtained from biochemical studies of the cervix.

Effects of brief postponement of the preovulatory LH surge on ovulation rates and embryo formation in eCG/prostaglandin-treated heifers

The aim of this study was to investigate whether prolongation of the period of preovulatory follicular development after superovulation reduces heterogeneity of oocytes of stimulated follicles with respect to the potential to mature, to ovulate, to be fertilized and to develop into embryos. Heifers were treated with eCG on Day 10 and prostaglandin (PG) 48 h later. At the time of eCG administration some of the heifers received a norgestomet implant (N) to suppress the LH surge. After 96 to 104 h, N was removed and an LH surge was induced with GnRH (G) (N/G); the other animals served as controls. Matured oocytes (Experiment A: n=9, 139 [N/G] and 11, 125 [Control] heifers, oocytes), zygotes and oviducts (Experiment B: n=8, 44 [N/G] and 9, 72 [Control] heifers, zygotes) and embryos (Experiment C: n=11, 205 [N/G] and 11, 165 [Control] heifers, embryos) were collected at 22 to 26 h, 38 to 52 h and 7 days after the LH surge, respectively. Hatched blastocyst formation of matured oocytes (Experiment A) was analyzed after 11 days of IVC after IVF. In vivo fertilization rate of zygotes, the presence of periodic acid-Schiff (PAS) positive granules in the oviduct (Experiment B) and stage of development of embryos (Experiment C) were analyzed stereomicroscopically. The mean interval between PG and the LH surge was 53.8+/-3 (SD) (N/G) vs. 42.4+/-4 h (Control). The maximum peripheral estradiol-17beta concentration (529+/-36 [SEM] [N/G] vs. 403+/-17 pmol/L [Control]) and the response to superovulation (25.4+/-2 [N/G] vs. 18.7+/-2 [Control]) were higher in N/G than in Control heifers. Hatched blastocyst formation rate (37.4 [N/G] vs. 33.6% [Control]), in vivo fertilization rate (69.0+/-14 [N/G] vs. 73.0+/-10% [Control]) and the yield of total embryos (3.8+/-1 [N/G] vs. 5.6+/-2 [Control]) did not differ between groups. The percentage of heifers with abundant PAS-positive granules in the distal ampulla (0 [N/G] vs. 31% [Control]) was reduced after N/G treatment. Prolongation of the period of preovulatory follicular development increased the number of mature follicles and ovulations but did not result in higher embryo yield, possibly because of an impaired oviductal environment.

Analysis of the economically optimal voluntary waiting period for first insemination

The voluntary waiting period (VWP) is defined as the time between parturition and the time at which the cow is first eligible for insemination. Determining the optimal VWP from field data is difficult and unlikely to happen. Therefore, a Monte-Carlo dynamic-stochastic simulation model was created to calculate the economic effects of different VWP. The model is dynamic and uses time steps of 1 wk to simulate the reproductive cycle (ovulation, estrous detection, and conception), the occurrence of postpartum disorders, and the lactation curve. Inputs of the model were chosen to reflect the situation of Dutch dairy cows. In the model, we initially created a cow of a randomly selected breed, parity, month of calving, calf status of last calving, and expected 305-d milk yield. The randomly varied variables were based upon relevant distributions and adjusted for cow statuses. The lactation curve was modeled by Woods function. The economic input values in the analysis included: cost of milk production (€0.07 to €0.20 per kg), calf price (€35 to €150 per calf), AI cost (€7 to €24 per AI), calving management cost (€137 to €167 per calving), and culling cost, expressed as the retention pay-off (€118 to €1,117). A partial budget approach was used to calculate the economic effect of varying the VWP from 7 to 15 wk postpartum, using a VWP of 6 wk as reference. Per iteration, the VWP with either the lowest economic loss or the maximum profit was determined as the optimal VWP. The optimal VWP of most cows (90%) was less than 10 wk. On average, every VWP longer than 6 wk gave economic losses. Longer VWP were in particular optimal for the first parity of breeds other than Holstein-Friesian, cows calving in winter with low milk production, high milk persistency, delayed peak milk yield time, a delayed time of first ovulation, or occurrence of a postpartum disorder, and while costs of milk production are low and costs for AI are high.

Effect of dose and day of treatment on uterine response to oxytocin in mares

To determine the effect of dose and day of oxytocin treatment on intrauterine pressure, 6 normal mares were treated with 10 or 25 IU oxytocin 2 days before ovulation, on the day of ovulation and 2 days after ovulation. Intrauterine pressure (IUP) was measured using micro-tip-catheters (one placed intrauterine, a second and third serving as reference sensors in the vagina and external to the mare) and transmitted by telemetry for 30 min to establish a baseline before saline was administered, iv, and for an additional 30 min after saline administration. Oxytocin was then given, iv, and IUP was recorded for 60 min. No change in IUP was observed after saline injection. The administration of both 10 (n=16) and 25 (n=10) IU oxytocin induced a response (P<0.01). The intensity of response depended on the day of administration (P<0.01) and the dose of oxytocin (P<0.001). The variation of response was significantly greater after 10 IU oxytocin (CV 15.78%) compared with 25 IU oxytocin (CV 6.42%). The uterine response was greatest on Day 2 prior to ovulation and lowest on Day 2 after ovulation. The response was negatively correlated to increasing plasma progesterone (10 IU oxytocin: r = -0.435, 25 IU oxytocin: r = -0.265). There was no correlation between the uterine response and plasma estradiol-17beta concentration (P<0.01). In conclusion the results of this study show that oxytocin administration to mares before ovulation provides a greater response than after ovulation. A decline in the intensity of response after ovulation can be compensated for with a higher dose of oxytocin. Furthermore, the use of the multiple catheter technique is an effective method for assessing changes in uterine pressure.

Characterisation of pregnancy losses after embryo transfer by measuring plasma progesterone and bovine pregnancy-associated glycoprotein-1 concentrations

The aim of this analysis was to determine whether pregnancy loss (PL) after embryo transfer (ET) in cattle was related to maternal progesterone (P4) concentrations during and shortly after ET, and maternal bovine pregnancy-associated glycoprotein-1 (bPAG-1) concentrations in plasma at days 25-35 of gestation. Embryos (n=260) were produced either in vivo after superovulation (n=115), or in vitro from oocytes (obtained with ovum pick-up) in co-culture (n=44) or cultured in a synthetic medium (n=101). Overall, PL was 56.9% (148) and no significant differences occurred in calving rate among the three embryo production groups. There was no difference in P4 concentrations on days 7-14 of gestation in the three groups, nor between ongoing and interrupted pregnancies. Between days 25 and 35 of pregnancy, bPAG-1 concentrations were unaffected by embryo production, but in cattle that had PL between days 26 and 120, four bPAG-1 profiles could be detected. Between days 25 and 32, bPAG-1 concentrations were influenced by PL, and concentrations were significantly lower in animals in which PL occurred between days 26 and 120 than in those animals that aborted later or calved at term. Early P4 concentrations suggested that maternal luteal factors were not responsible for PL which appeared to be caused by impaired conceptus development (regardless of embryo type) as reflected by low maternal bPAG-1 concentrations prior to embryonic death.

Effect of milk yield characteristics, breed, and parity on success of the first insemination in Dutch dairy cows

The objective of this study was to determine the contribution of cow factors to the probability of a successful first insemination (SFI). The investigation was performed with 51,791 lactations from 1,396 herds obtained from the Dutch dairy cow database of the Cattle Improvement Co-operative (CRV). Cows that had the first insemination (AI) between 40 and 150 d postpartum were selected. The first AI was classified as successful when cows were not reinseminated and either calved between 267 and 295 d later or were culled within 135 to 295 d after first AI. The lactation curve characteristics of individual lactations were estimated by Wilminks curve using the test-day milk records from CRV. The lactation curve characteristics (peak milk yield, milk yield at the first-AI date, time of peak yield (PT), and milk persistency) were calculated. Breed, parity, interval from calving to first AI (CFI), lactation curve characteristics, milk production traits, moment of AI related to PT (before or after PT), calf status, month of AI, and month of calving were selected as independent variables for a model with SFI as a dependent variable. A multivariable logistic regression model was used with farm as a random effect. Overall SFI was 44%. The effect of parity on SFI depended on CFI. The first-parity cows had the greatest SFI (0.43) compared with other parities (0.32-0.39) at the same period of CFI before 60 d in milk (DIM), and cows in parity ≥5 had the least SFI (0.38-0.40) when AI was after 60 DIM. After 60 DIM, extending CFI did not improve SFI in the first-parity cows, but SFI was improved in multiparous cows. Holstein-Friesian cows had lesser SFI (0.37) compared with cross-breed cows (0.39-0.46). Twin and stillbirth calving reduced SFI (0.39) compared with a single female calf (0.45) or a male calf (0.43) calving. The SFI in different months of AI varied and depended on CFI. Cows that received AI before 60 DIM had a lesser SFI, especially in March, June, and July (0.18, 0.35, and 0.34, respectively). Artificial insemination before PT reduced SFI (0.39) in comparison with AI after PT (0.44). The effect of milk yield at the first-AI date on SFI varied depending on CFI. After 60 DIM at the same period of CFI, a high level of milk yield at the first-AI date reduced SFI. In conclusion, knowledge of the contribution of cow factors on SFI can be applied to support decision making on the moment of insemination of an individual cow in estrus.