Promotion of ovarian follicular development by injecting vascular endothelial growth factor (VEGF) and growth differentiation factor 9 (GDF-9) genes

Ovarian follicular development in mammals is the complex process including endocrine, paracrine and autocrine. There is the development of four basic stages of ovarian follicles, i.e. the primordial, primary, secondary and tertiary or Graafian follicles. There are few blood vessels in the cortical area where primordial and primary follicles are assembled. The development of these follicles is stimulated by oocytes derived factor including growth differentiation factor 9 (GDF-9) or bone morphogenetic protein 15 (BMP-15). Porcine GDF-9 complementary DNA (cDNA) cloned, and then injected its gene into the ovary in gilts. The injection of porcine GDF-9 gene resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles, indicating that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary. On the other hand, the development of antral follicles is associated with increased density of blood vessels within the theca cell layers surrounding the follicles. A recent study reported that vascular endothelial growth factor (VEGF) play an important role in the process of thecal angiogenesis during follicular development. To investigate whether additional induction of thecal angiogenesis would support subsequent follicular development, miniature gilts were directly injected VEGF gene into the ovary. Injection of VEGF gene increased the levels of mRNA expression of VEGF 120 and VEGF 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene compared with those treated with eCG alone, indicating that the regulation of thecal angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. This technique may be an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.     

Timing of preovulatory LH surge and ovulation in superovulated sheep are affected by follicular status at start of the FSH treatment.

The aims of this study were to evaluate the chronology of periovulatory events (oestrus behaviour, LH surge and ovulation) in 16 superovulated Manchega sheep and to determine whether follicular status at start of the FSH supply might affect their occurrence. Mean timing for onset of oestrus behaviour was detected at 28.1 +/- 0.7 h after sponge withdrawal; the preovulatory LH surge and ovulation started at 37.2 +/- 0.7 h and 65.4 +/- 0.7 h after progestagen withdrawal, respectively. The intervals between oestrus, LH surge and ovulation were affected by a high individual variability, which might be the cause for reported decreased efficiency in embryo production. Current results also addressed the role of follicular status at start of the superovulatory treatment on the preovulatory LH surge and the ovulation. The interval LH surge-ovulation was increased in ewes with a growing dominant follicle at starting the FSH treatment (32.3 +/- 0.9 vs 28.6 +/- 0.5 h, p < 0.05). The developmental stage of the largest follicle at starting the superovulatory treatment also affected occurrence of LH surge and ovulation; follicles in growing phase advanced the occurrence of the LH surge and ovulation when compared to decreasing follicles (33.0 +/- 1.0 vs 43.5 +/- 1.1 h, p < 0.05, for LH peak and 60.7 +/- 1.1 vs 72.8 +/- 1.2 h, p < 0.05, for ovulation). Thus, only ewes with growing follicles ovulated prior to 55 h after sponge withdrawal; conversely, no sheep with decreasing follicles ovulated earlier than 67 h, when an 85.7% of the ewes bearing growing follicles has ovulated at 63 h.     

In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes

To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.     

Ovarian follicular cysts in dairy cows: an abnormality in folliculogenesis

Ovarian follicular cysts are a major reproductive problem in lactating dairy cows. The primary physiological defect leading to the formation of ovarian follicular cysts is a failure of the hypothalamus to trigger the preovulatory surge of luteinizing hormone (LH) in response to estradiol. The factor responsible for this hypothalamic defect may be progesterone. Intermediate levels of progesterone have been shown to prevent ovulation and promote persistence of dominant follicles in normal cycling cows. Recently, we found that 66% of cows with ovarian follicular cysts had progesterone concentrations in an unusual, intermediate range (0.1-1.0 ng/mL) at the time of their detection. A majority of new follicles (76%) that develop in the presence of these intermediate progesterone concentrations became cysts. Only 10% ovulated. Based on these observations, a novel model for the formation and turnover of ovarian follicular cysts is proposed.     

Periovulatory endocrinology and oocyte maturation in unmated mature blue fox vixens (Alopex lagopus)

Nine of 10 mature blue fox vixens (Alopex lagopus) in spontaneous oestrus ovulated approximately 2 days after the preovulatory increase in luteinizing hormone (LH). Plasma concentrations of follicle-stimulating hormone and progesterone increased simultaneously with the LH peak, whereas oestradiol-17 beta peaked 1 day previously. In the tenth vixen, an LH peak was not observed, and neither visible follicles nor corpora lutea were found in the ovaries 6 days after peak vaginal electrical resistance. Eggs were ovulated as primary oocytes, but oocyte maturation was initiated within the day of ovulation (2 days after the LH peak). Within the next 2 days (3-4 days after the LH peak) the first polar body was extruded, and the cumulus mass was completely dissociated from the zona pellucida. The interval between the preovulatory LH peak and initiation of the final oocyte maturation is thus considerably longer in the blue fox than for example in the cow (48-72 h compared with 9-12 h). This suggests that the relationship between these two events is somewhat different in the blue fox.     

Strategies for Using eFSH for Superovulating Mares

The standard treatment for superovulation of mares is to administer equine follicle-stimulating hormone (eFSH) for 4 to 5 days to stimulate multiple follicles and human chorionic gonadotropin (hCG) to induce synchronous ovulations. Objectives of this study were: (1) to determine whether a short-term (3-day) eFSH treatment protocol would result in similar ovulation and embryo recovery rates compared with the standard eFSH protocol; (2) to determine the efficacy of a decreasing dose of eFSH (step-down protocol) on ovulation rate and embryo recovery; (3) to compare the efficacy of hCG and recombinant equine luteinizing hormone (reLH) for inducing ovulation in FSH-treated mares; and (4) to compare embryo recovery rates and embryo size when mares are flushed at 6.5 or 7.0 days after ovulation. Forty light-horse mares were used in 2005 (experiment 1) and 20 different mares were used in 2006 (experiment 2). In experiment 1, mares were randomly assigned to one of three treatment groups: (1) untreated controls, (2) standard eFSH treatment (12.5 mg intramuscularly twice daily), and (3) 3-day eFSH treatment. In experiment 2, mares were randomly assigned to one of four treatments: (1) untreated controls, (2) standard eFSH protocol, (3) 3-day eFSH treatment, and (4) step-down eFSH treatment (12.5 mg twice daily day 1, 8.0 mg twice daily day 2, 4.0 mg twice daily day 3). Within each treatment, mares were given either hCG (2,500 IU) or equine LH (750 mg, EquiPure LH; reLH) to induce synchronized ovulations. Embryo recovery was performed either 6.5 or 7.0 days after ovulation. In experiment 1, numbers of preovulatory follicles and ovulations were less for mares in the 3-day treatment group than the standard group, but were greater than for controls. Embryo recovery per flush was higher in the standard group (2.6) than the 3-day eFSH treatment (0.8) or control groups (0.8). In experiment 2, the number of preovulatory follicles and number of ovulations were greater in the standard and 3-day treatment groups than in control and step-down groups. The percent embryo recovery per ovulation and mean embryo grade were similar for all groups; however, the embryo recovery per flush was higher for mares in the standard treatment than controls (1.3 vs 0.6) but was similar to the 3-day (1.1) and step-down (0.8) treatments. Embryo recovery was similar for flushes performed on days 6.5 and 7.0 post-ovulation. The percentage of control mares ovulating within 48 hours in response to hCG or reLH was similar. In contrast, a higher percentage of eFSH-treated mares ovulated within 48 hours in response to reLH than hCG (92% vs 71%). In both years, the 3-day eFSH treatment protocol resulted in a greater number of preovulatory follicles and a greater number of ovulations than untreated controls. Unfortunately, the increased ovulation rate for mares administered eFSH for 3 days did not result in a greater number of embryos recovered per flush in either year. Use of a step-down eFSH treatment protocol resulted in fewer preovulatory follicles, fewer ovulations, and fewer embryos as compared with the standard eFSH treatment. In conclusion, the standard eFSH treatment resulted in a greater embryo recovery rate per cycle than either the 3-day or step-down treatment protocols. Recombinant equine LH was more effective than hCG in causing ovulation in eFSH-treated mares.     

The effect of pretreatment with different doses of GnRH to synchronize follicular wave on superstimulation of follicular growth in dairy cattle

The objective of this study was to evaluate the efficiency of gonadotropin releasing hormone (GnRH) and GnRH doses in synchronizing follicular wave emergence as a pretreatment for superovulation in cattle. Fourteen Holstein-Friesian cows 6 days from estrus were randomly assigned to receive 100 microg (n=4), 50 microg (n=5), or 25 microg (n=5) of GnRH. Superovulation was induced with injections of porcine FSH (pFSH) twice daily, decreasing the dose (total 42 AU) over 5 days beginning 2.5 days after receiving GnRH. On the 7th and 8th injections of pFSH, 750 microg of PGF(2alpha) was also given. With the exception of one cow that was given 50 microg of GnRH, ovulation was induced in all cows from the three groups and the new follicular wave emergence was observed. The total number of follicles for the 25 microg GnRH group was less than that observed for the 100 microg GnRH group (P<0.05), although there were no differences between the 100 microg, 50 microg and 25 microg GnRH groups with respect to the number of preovulatory follicles (>or=10 mm) and CL. The numbers of normal embryos were greater for the 25 microg GnRH group than the 100 or 50 microg GnRH groups (P<0.01); however, the numbers of ova/embryos did not differ significantly between the three groups. These results suggest that 25 microg of GnRH was sufficient to induce ovulation and follicular wave emergence. On day 6 of the estrous cycle, a reduction of the dose of GnRH to synchronize follicular wave emergence as a pretreatment for superstimulation promotes transferable embryos.     

Passive immunoneutralization of endogenous inhibin increases ovulation rate in miniature Shiba goats

We reported previously that passive immunization against inhibin enhances follicular growth and increases the ovulation rate. However, the ovulation rate was not comparable to the number of follicles. Therefore, the aim of this study was to attempt to increase the ovulation rate by increasing the interval between inhibin immunization and PGF2alpha injection. Five miniature Shiba goats were treated with 10 ml inhibin antiserum (inhibin-AS) developed against [Tyro30]-inhibin alpha (1-30). A control group (n=5) was treated with normal goat serum. All animals were injected intramuscularly with 125 microg PGF2alpha 72 h after treatment to induce estrus and ovulation. Blood samples were collected for hormonal assay and the ovulation rate was determined by laparotomy. In contrast to the control group, there was a significant increase in plasma concentrations of FSH in the immunized group. After luteolysis, plasma concentrations of estradiol-17beta increased markedly to a preovulatory peak about 2 folds higher (P<0.01) than that of controls. In addition, the ovulation rate was greater in the immunized group (14.4 +/- 2.2) than in the control group (2.2 +/- 0.6), and the mean number of follicles > or = 4 mm in diameter was 10.0 +/- 0.8 in the inhibin-AS group compared with 2.4 +/- 0.3 in control group. The present results demonstrate that immunoneutralization of endogenous inhibin increased FSH secretions in miniature shiba goats. The increased FSH secretion enhanced follicular growth and increased the ovulation rate. Additionally, increasing the interval between inhibin-AS and PGF2alpha injections (to 72 h) resulted in a greater ovulation rate compared with the previous protocol (48 h). Therefore, inhibin-AS treatment proved to be an effective alternative to exogenous gonadotropin methods for induction of superovulation in goats.     

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.     

Quantitative Echotexture Analysis for Prediction of Ovulation in Mares

The aim of this study was to predict the ovulation in mares by quantitative analysis of the echotextural changes of preovulatory follicular walls. Four mares of breeding age with 32 preovulatory follicles and 11 anovulatory follicles were observed by ultrasonography. The slope of the regression line of the follicular wall and the echogenicity score of granulosa layer (GL) and anechoic layer (AL) were measured from the images on Days -3 (Day 0 = ovulation), -2, and -1, respectively. GL was scored from 1 (anechoic) to 3 (echoic), and prominence of AL was recorded from 1 (gray and thin) to 3 (black and thick). The results indicated that the regression line of the follicular wall for 81.3% (26/32) of preovulatory follicles had the slope value ≥19.0 on Day -1, in which 4 of the 26 preovulatory follicles were ≥19.0 on Day -2 already. Mean slope value on Day -1 (21.9 ± 1.5) was significantly greater (P < .01) than on Day -2 (15.0 ± 1.4) and Day -3 (14.0 ± 1.1). All of the slope values for the 11 anovulatory follicles were <19.0 on any given day. GL and AL scores of preovulatory follicles were significantly greater (P < .01) than in anovulatory follicles on Days -3, -2 and -1; nevertheless, only 28.1% (9/32) of preovulatory follicles scored 3 for both GL and AL simultaneously on Day -1. All anovulatory follicles scored <2 for both GL and AL on Day -1. It was concluded that the slope of the regression line of the follicular wall is useful in predicting preovulatory follicles within 48 hours of ovulation when the value is ≥19.0. Of these follicles (N = 26), 84.6% (22/26) were predicted to ovulate within 24 hours, and 15.4% (4/26) within 24 to 48 hours.

Introduction

Insemination in mares by accurately predicting the time of ovulation may obtain maximum fertility with minimum use of semen, and therefore would definitely be a profitable advantage in the horse farming business. The optimal time for insemination with frozen-thawed semen usually include a shorter interval than if fresh semen or natural breeding is used. To achieve the maximal pregnancy rates with frozen-thawed semen, it is necessary to inseminate mares during a period between 12 hours pre- and 6 hours post-ovulation.[1] Therefore, if the timing of ovulation could be predicted, it would be helpful for the veterinarian to inseminate a mare only once per cycle if performed very close to the time of ovulation. [2] In recent years, many indicators have been reported for predicting impending ovulation in mares, including measurement of electrical resistance of the vaginal mucus, [3] the distinguishable endometrial folding pattern of uterus in estrus, [4] changes in size and shape of the preovulatory follicles, [5, 6 and 7] and the echotexture changes in the preovulatory follicular wall. [8] The latter has been more efficient for predicting the imminence of ovulation; nevertheless, their assessment of criterions was scored subjectively. The hypothesis for this study was based on the published report from Gastal et al in 1998 [8]; they found that 2 echotexture changes of the preovulatory follicle-increasing echogenicity of the granulosa layer and increasing prominence of an anechoic layer beneath the granulosa, were detected in the follicular wall as ovulation approached in mares. Computer-assisted image analysis is an advanced technology for diagnostic ultrasonography to improve the reproductive management of patients. [9, 10 and 11] The purpose of this study is to quantify the echotextural changes in the preovulatory follicular wall as ovulation approaches using computer-assisted image analysis, so that the quantified echotexture changes could serve as an indicator for prediction of ovulation in mares.

Materials and Methods

Animals and Ultrasonography

Four non-lactating and nonpregnant mixed mares between 4 and 14 years of age and weighing between 450 and 550 kg were studied from January to December 2001. The geographic area of the mares in this study was in subtropical Taiwan of the northern hemisphere. All mares were maintained on alfalfa/grass hay and had access to water and mineralized salt. A teaser stallion was introduced to detect the estrus signs of mares about 2 weeks after the end of the last estrus. Follicular changes were monitored with a real-time B-mode linear assay ultrasound scanner, equipped with a 7.5-MHz transrectal probe (Model Scanner 200 Vet, Pie Medical, The Netherlands). Upon detection of a preovulatory follicle, ultrasound examination was performed daily and continued until ovulation. A total of 32 preovulatory follicles and 11 anovulatory follicles were identified from a retrospective determination.Ultrasonographic images were recorded on Hi-8 MP videotape with a Sony DCR-TRV 120 Digital-8 camera. The brightness and contrast controls of the monitor and the time-gain compensation of the scanner were standardized to constant settings throughout the observation period.

Image Analysis

Still images were subsequently captured and saved as TIF files by computer using a digital image analysis program (Image-Pro Express V4.0 for Windows, Media Cybernetics, L.P., USA) with a resolution of 640 × 480 pixels and 256 shades of gray. Echotexture of the regions of interest was defined in terms of pixel intensity ranging from 0 (black) to 255 (white). Three ultrasonographic images of each preovulatory follicle at its distinctly discernible cross section were subsequently selected. To avoid the enhancement of through-transmission, sampling regions were located within the 10 or 2 o'clock position for measurement of pixel values (Fig 1). The pixel values were measured with the “Line Profile” tool, which involved sampling pixel values along a line traversing the follicle wall from the peripheral antrum, GL, AL, to the stroma. A graph of the pixel intensities along the line was produced ( Fig 2). The GL was defined as the highest pixel after which there was a sequential fall in gray-scale values. The pixel values along the curve (P0, P1, P2) were obtained as an average of 9 measurements (3 images per follicle and 3 lines per image) and were used to measure the slope of a regression line of the fall segment ( Fig 2).     

Follicular development after ovum pick-up and fertilizability of retrieved oocytes in postpartum dairy cattle

This study aimed to evaluate gonadotropin secretion and the developmental competence of follicular oocytes in dairy cattle during the early postpartum (PP) period. The number of follicles developed after transvaginal ultrasound-guided ovum pick-up (OPU) and fertilizability of retrieved oocytes were compared between cows in which the first dominant follicle (DF) ovulated (ovulated group, n=4) and did not ovulate (non-ovulated group, n=3), and between early PP (early PP group, n=2) and after the resumption of the estrous cycle (cyclic group, n=2). Follicular ablation was performed 2-4 days after the detection of DF in the second follicular wave PP. OPU was repeated 3-5 times at 3 or 4-day intervals from 3-4 days after the follicular ablation. At OPU, the follicles were enumerated and all those > or = 5 mm in diameter were aspirated. Recovered oocytes were subjected to in vitro maturation and fertilization. Both criteria were similar between ovulated and non-ovulated groups, and between early PP and cyclic groups. These results suggest that FSH/LH secretions required for follicle recruitment and subsequent follicular growth during the early PP period are similar to those after resumption of the estrous cycle. They also indicate that follicular oocytes during the early PP period have developmental competence.     

Ultrasonographic characteristics of the preovulatory follicle preceding and during ovulation in mares

Changes in appearance of preovulatory follicles were observed with real-time ultrasonography prior to and during ovulation in mares. Preovulatory follicles of 15 mares were scanned at < 1 hr intervals for 12 hr or more frequently if displaying signs of impending ovulation. If ovulation was not imminent at the end of 12 hr (n = 2), mares were removed from the trial. Mean follicular diameter decreased 13% from 30 minutes prior to ovulation until the beginning of ovulation. Fifteen to 77 minutes (mean = 41 min) prior to ovulation, a break in or a protrusion of the follicular wall toward the ovulation fossa was visualized in all follicles and was a consistent indicator of impending ovulation. A rapid decrease in size of follicles (ovulation) occurred within a period of 5 to 90 seconds (mean = 42 sec). Little or no fluid remained in the antrum following ovulation. An increase in echogenicity (whiteness) of the follicular wall and echogenic “spots” within the follicle were frequently visualized (13/13, 100% and 7/13, 54% respectively) prior to ovulation; however, prediction of time of ovulation could not be based solely on these individual changes.     

Ovulation of the preovulatory follicle originating from the first-wave dominant follicle leads to formation of an active corpus luteum

The objective of our study was to compare the characteristics of the corpus luteum (CL) formed after ovulation of the dominant follicle (DF) of the first follicular wave (W1) and those of the CL formed after ovulation of the DF of the second (induced) follicular wave (W2). Non-lactating Holstein cows were used for this study. In Experiment 1, cows were treated with PGF2α and GnRH on days 6 and 8 (day 0 = day of follicular wave emergence) for W1 (n = 6) and W2 (n = 6), respectively. Dominant follicles were aspirated on day 9 to quantify the amounts of mRNA (VEGF120, VEGF164, FGF-2, StAR, P450-scc and 3β-HSD) in granulosa cells (GC). In Experiment 2, the size and blood flow area of the CL formed after ovulation of the DF in W1 (W1CL; n = 6) and W2 (W2CL; n = 6) (the day of DF ovulation in W1 and W2 was day 10) were evaluated on days 12, 15, 18 and 21. The plasma P4 concentration was measured on days 10 to 21. The amounts of VEGF164, P450-scc and 3β-HSD mRNA were higher (P < 0.05) in the DF in W1, and those of VEGF120,FGF-2 and StAR mRNA tended to be higher (P < 0.1) in the DF in W1. The size of the CL was greater in the W1CL on days 15, 18 and 21. The blood flow area of the CL was greater in the W1CL on days 12 and 15. The plasma P4 concentrations were higher in the W1CL. These results indicate that the CL formed after ovulation of the DF in W1 was greater in terms of size, blood flow and plasma P4 concentration.     

Induction of ovarian cystic follicles in sheep

Cystic follicles are a significant cause of infertility in women, dairy cattle and sheep. Sheep were used as a model to identify factors that may elicit formation of cystic follicles. Insulin resistance and elevated LH activity were tested in overweight ewes because of associations among these factors and the formation of cystic follicles. Sheep were synchronized using a progesterone-releasing pessary and insulin resistance was induced during the synchronization period through administration of bovine somatotropin. Following removal of pessaries follicular growth was stimulated by treatment with eCG or eCG and hCG (PG-600). Follicular growth was monitored via daily transrectal ultrasonography and blood samples were collected for hormonal analyses. Six of 18 ewes had a subnormal or absent preovulatory gonadotropin surge and developed cystic follicles. Neither insulin resistance nor elevated LH activity were associated with formation of cystic follicles. Ewes that developed cystic follicles were heavier (93 +/- 4 kg) than ewes that ovulated (81 +/- 3 kg; P = 0.02). Furthermore, following pessary removal and initiation of daily ultrasonography, ewes that developed cystic follicles lost body weight (-3 +/- 1%), while ovulatory ewes continued to gain body weight (1 +/- 1%; P = 0.005). It is speculated that in heavy ewes metabolic factors associated with acute body weight loss inhibit the positive feedback of estradiol and thereby suppress the preovulatory gonadotropin surge leading to formation of cystic follicles.     

Glutathione content of in vivo and in vitro matured canine oocytes collected from different reproductive stages

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.     

Observations on the influence of exogenous gonadotrophins and cloprostenol on ovulation in gilts

We studied the effects of gonadotrophins and prostaglandin (PG) F_2α on ovulation in gilts. Twenty-eight gilts were induced to ovulate using 750 IU pregnant mares serum gonadotrophin (PMSG) and 500 IU human chorionic gonadotrophin (hCG), administered 72 h apart. At 34 and 36 h after hCG, gilts received injections of either 500 μg or 175 μg PGF_2α (cloprostenol), or had no injections. Laparotomies were performed at 36 h (cloprostenol gilts) or 38 h (controls) after hCG injection. The ovaries were examined and the proportion of preovulatory follicles that had ovulated (ovulation percent) was determined at 30 min intervals for up to 6 h. The number of gilts in which ovulation was initiated and the ovulation percent increased (p<0.001) with time, but was not affected by treatment. Many medium sized follicles (≤6 mm) were also observed to ovulate, or to exhibit progressive luteinization without overt ovulation, during the surgical period. A discrepancy between numbers of preovulatory follicles and corpora lutea suggests that luteal counts may not be an accurate assessment of ovulation rate following gonadotrophic stimulation.     

Follicle population dynamics in sheep with different ovulation rate potentialsDynamique folliculaire terminale chez des ovins de potentiel ovulatoire variéVeränderungen der follikelzahl in schafpopulationen mit unterschiedlich hoher ovulationsrate

To investigate the factors contributing to the different ovulation rates observed in two strains of sheep (Booroola 5.2, Merino, 1.2) and after immunization against androstenedione (Immunized 1.8, Control 1.3), in vivo monitoring of follicular kinetics followed by histological examination of the ovaries was performed during the late luteal and follicular phases. Ewes had the three largest follicles of each ovary ink-labelled at days 13 and 15 and were ovariectomized after the beginning of oestrus.High ovulation rate was not associated with a more numerous antral follicle population in either Booroola ewes or immunized ewes. Furthermore, in Booroola ewes (r = 0.22) and in immunized ewes (r = ?0.02), there was no correlation between the number of antral follicles per ovary and the ovulation rate.The reasons for the high ovulation rate became clear when preovulatory enlargement was followed by ink-labelling. An extended period of time during which recruitment of ovulatory follicles takes place, together with a low incidence of selection through atresia and the ability of fully grown follicles to wait for ovulation are the features involved in the high ovulation rate of the Booroola ewes. A lower incidence of selection together with an unaltered recruitment leads to the increased ovulation rate noticed after immunization.     

Relationship between oocyte maturation and fertilization on zygotic diversity in swine

Two experiments were conducted to examine how oocyte maturation and fertilization influence zygotic diversity in swine. In the first experiment, the distribution of oocyte maturation was compared to that of zygotic development. Oocytes were aspirated from follicles of 31 gilts and classified into stages of meiosis. Zygotes were flushed from oviducts of 19 additional gilts and classified into stages of meiosis and fertilization. The second experiment examined whether the time from ovulation to fertilization was constant among all oocytes. To test this premise, four to six oocytes from follicles of 10 mated gilts were aspirated just before or during ovulation, stained and transferred back into the oviducts of these same gilts. Zygotes were recovered 10 h later to determine whether the first oocytes ovulated were the more developed zygotes and, conversely, whether the last oocytes to be ovulated represented the lesser developed contemporaries. The skewed (P less than .05) distribution of oocyte maturation was similar to that of zygotic development. Regression of the frequency distribution describing early oocyte maturation resulted in a line with a slope (.59) that was similar to the slope (.58) of the regressed distribution of zygotic development. Likewise, the order of ovulation and order of subsequent stages of zygotic development were similar. These data suggest that variation in zygotic development in swine was due to variability in oogenesis; the time from ovulation to fertilization appeared to be constant.