Evaluation of three classification methods of antral follicle count and fertility to the timed artificial insemination in cattle

The controversial data about antral follicle count (AFC) may be partially explained by the different criteria used to determine what is high, intermediate and low AFC. This study evaluated different classification methods for AFC groups, relating them to the conception rate, dominant follicle size and body condition score (BCS) in cows submitted to timed artificial insemination (TAI). Nelore cows (Bos indicus; n = 935), received a reproductive program consisting of TAI and natural breeding. Conception rate, BCS and dominant follicle size during TAI were evaluated by three AFC methodologies: i) mean and standard deviation: low (≤ 15 follicles); intermediate (≥ 16 to ≤ 44 follicles) or high (≥ 45 follicles); ii) quartiles: low (≤ 15 follicles); intermediate (≥ 16 to ≤ 39 follicles), or high (≥ 40 follicles); and iii) AFC score: I (low; ≤ 15 follicles); II (intermediate; ≥ 16 to ≤ 30 follicles); III (high; ≥ 31 to ≤ 44 follicles) or IV (very high; ≥ 45 follicles). Data were analyzed by a GLIMMIX and Tukey test or binary logistic regression model (P ≤ 0.05). The conception rate to TAI was influenced (P < 0.05) by AFC in the three methods classification, being the highest conception rate observed in the low AFC group regardless of method utilized: Mean (low 61.73%^a, intermediate 54.02%^ab and high 49.48%^b), Quartiles (low 61.73%^a, intermediate 53.59%^ab and 51.46%^b) and Score (I 61.73%^a, II 54.80%^ab, III 53.23%^ab and IV 49.48%^b). There were variations (P < 0.05) in the conception rate within the 2.50 to 2.75 BCS range for all AFC classification methods, with the low AFC females presenting the best results, regardless of the method used. Also, females with low AFC showed larger (P < 0.05) diameters of dominant follicles at the TAI regardless of method. The different methodologies used (Mean, Quartile and Score) to AFC classification showed a consistency between the main findings, and we believe that this standardization will facilitate the interpretation of data involving AFC.     

Effect of follicle cells on in vitro fertilization of pig follicular oocytes

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.     

Effects of prolonging administration gonadotropin on unexpectedly poor ovarian responders undergoing in vitro fertilization

Background

There are still some patients who show poor response to ovarian stimulation prior to evidence of normal ovarian reserve in vitro fertilization. However, there are few studies about how to treat the unexpectedly ovarian poor responder in vitro fertilization. The main aim of this study evaluate the effect of prolonging administration follicle-stimulating hormone in woman with the unexpectedly ovarian poor responder in vitro fertilization on implantation rate, clinical pregnancy rate and live birth rate.

Methods

922 patients subjected to IVF were divided into two groups according to the predicted criterion of ovarian poor response. 116 patients predicted poor response received the short protocol (group C). The others received the long protocol, among the latter, there were 149 patients undergoing unexpectedly ovarian poor response (group B) and 657 patients exhibited normal ovarian response (group A). The doses of gonadotropin, duration of administration, implantation rate, clinical pregnancy rate and live birth rate were recorded among three groups.

Results

The implantation rate of embryo, clinic pregnancy rate and delivery rate are similar between the group A and group B, while there are significant differences between the doses of gonadotropins (35.1 +/- 8.9 ampules vs.53.0 +/- 15.9 ampules) and the duration of administration (15.3 +/- 3.6D vs. 9.8 +/- 2.6D) of these two groups. There are no significant differences about clinical pregnancy rate and live birth rate between group B and group C.

Conclusion

Prolonging administration gonadotropin on the unexpectedly poor ovarian responders does not lower live birth rate in vitro fertilization.     

Ovarian follicle growth and consequences for fertility in sheep

Understanding the pattern of ovarian follicle development is seen as an important step leading to the development of techniques that maximise fertility in sheep. Repeated observations of the growth of individual follicles have led to the understanding that follicles develop in a wave-like pattern during the oestrous cycle, with two to four waves per cycle being the most common. The ease with which follicle waves are described seems to depend on the their frequency and the number of follicles per wave. There is evidence for the largest follicle(s) of a follicle wave inhibiting the development of other follicles; however, in some cases this is not apparent as other follicle waves emerge when a previous large, healthy follicle is still present. Follicle development can be manipulated using exogenous gonadotrophins or progestagens and these have been shown to alter the number or age profile of developing follicles. The ovulation of aged follicles in cattle clearly has a detrimental effect on fertility, but this relationship is less clear and seems to be less critical in sheep. Recent findings at the molecular level show that the bone morphogenetic proteins (BMP) and their receptors are critically involved in the control of ovulation rate, but fully understanding their mechanism remains to be described. This highlights the potential for the integration of molecular and physiological findings to better develop methods to manipulate follicle development and reproduction in sheep.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Localization of nucleoside phosphatases in the antral follicle of the guinea pig ovary

Ultrastructural localization of nucleozidphosphatases (5'-nucleotidase, adenosin triphosphatase (ATPase) and beta-glicerophosphatase) in antral follicles of the guinea-pig ovary has been studied. Certain heterogeneity has been found in distribution of the enzymes: the cells in the follicular tunic possess the greatest 5'-nucleotidase and ATPase activity. When 5'-adenosin monophosphate (5'-AMP) is used as a substrate, the lead phosphate residue is mainly revealed in the external surface of plasmolemma and as "caps" in the margical zone of nucleoplasm. ATPase activity is chiefly observed in nucleoli of granular cells and in those of the external follicular tunic cells. Histochemical reaction with 5'-AMP proceeds most intensively in the lucid tunic and in processes of the granular cells contacting with the oocyte. A possibility is discussed on participation of the metabolic enzymes, that localize in these structures, in the mechanisms controlling the oocyte maturation.     

In vitro fertilization and development of cumulus oocytes complexes collected by ultrasound-guided follicle aspiration in superstimulated llamas

The objective was to evaluate the developmental competence of cumulus-oocyte complexes (COC) collected by follicular aspiration in llamas treated with FSH or eCG. Llamas were assigned randomly to two groups (n = 16 per group) and treated, at the time of ovarian follicular wave emergence, with either: 1) 25 mg of FSH im, twice daily for 4 d; or 2) 1000 IU of eCG as a single i.m. dose. The start of gonadotropin treatment was considered Day 0. Both groups were given 5 mg of Armour Standard LH im on Day 6, and COC were collected by follicle aspiration on Day 7. Expanded COC collected from FSH- (n = 157) and eCG-treated llamas (n = 151) were fertilized in vitro using epididymal sperm, and presumptive zygotes were in vitro cultured in SOF medium for 8 d. The FSH and eCG treatment groups did not differ with respect to: the number of follicles ≥7 mm (16.0 ± 2.7 vs 14.0 ± 1.9, respectively; P = 0.5); the number of COC collected (11.5 ± 1.9 vs 9.7 ± 1.2; P = 0.4); the number of expanded COC (9.8 ± 1.4 vs 9.4 ± 1.2; P = 0.8); or the percentage of presumptive zygotes which developed into 2 to 8 cell stage embryos (65.3 vs 63.1), morulas (46.2 vs 42.5), or blastocysts (23.1 vs 20.5; P > 0.05). In conclusion, FSH and eCG treatments were equally effective for recovery of a high number of expanded COC which were used directly for in vitro fertilization. Furthermore, rate of embryo development was not significantly affected by the gonadotropin treatment used.     

Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro

The role of follicle cells in the acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eight-cell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.     

Effects of gonadotropin treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of oocytes in prepubertal gilts

This study was undertaken to compare the effects of FSH-pituitary (FSH-P), eCG, and a combination of gonadotropins containing 400 IU eCG and 200 IU hCG (PG 600) on the growth of large follicles, oocyte quality and in vitro fertilization (IVF) rate of in vitro matured (IVM) oocytes in prepubertal gilts. The ovaries were removed via midventral laparotomy 48 h (Experiment 1) or 72 h (Experiment 2) after the first injection. In Experiment 1, 30 gilts received 1 of 5 treatments: 1) saline (3 ml i.m., once, n = 6); 2) FSH-P8 (8 mg i.m., twice, with a 24-h interval, n = 6); 3) FSH-P16 (16 mg i.m., twice, with a 24-h interval, n = 6; 4) eCG (1000 IU i.m., once, n = 6); or 5) PG 600 (5 ml i.m., once, n = 6). Compared with saline, treatment with PG 600 or eCG induced significant (P < 0.05) growth of large follicles (> or = 6 mm). In Experiment 2, 16 gilts received 1 of 5 treatments: 1) saline (n = 4); 2) FSH-P8 (n = 4); 3) FSH-P16 (n = 4); 4) eCG (n = 4), or 5) PG 600 (n = 4). The same injection protocol as in Experiment 1 was used. Compared with treatment with FSH-P8 or FSH-P16, eCG increased (P<0.05) the number of large follicles. The proportion of good oocytes was increased (P<0.05) with FSH-P8 or FSH-P16 compared with treatment with eCG or PG 600. Moreover, oocytes from eCG-treated gilts had a greater (P<0.05) rate of male and female pronuclei than FSH-P or saline-treated gilts. In conclusion, treatment with FSH-P resulted in a higher proportion of oocytes with multilayer cumulus cells, whereas treatment with eCG resulted in higher pronuclear rates following in vitro fertilization in prepubertal gilts.     

Platelet count and mean platelet volume in an Algerian population indicating a low prevalence of Mediterranean macrothrombocytopenia

Platelet count was 225 X 10(9)/l in 225 healthy male Algerians and 263 X 10(9)/l in 208 females. The mean platelet volume was 9.15 fl in the males and 9.30 in the females. The figures agree with those obtained in a British and an American population, but differed from those of an Australian population of immigrants from Mediterranean countries, essentially Italy and Greece. The prevalence of Mediterranean thrombocytopenia must therefore be low in Algeria.     

Indications for in vitro fertilization and embryo transfer

The main indications of IVF-ET, i.e. tubal sterility, idiopathic infertility and male infertility are critically discussed. Tubal sterility is in our opinion the only clear clinical indication. The IVF-ET application in cases of idiopathic infertility can be considered as an empirical therapeutic approach. According to our own results and those of other authors, male infertility is the most questionable indication to IVF-ET.     

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

Effect of exogenous gonadotrophin (PMSG) on the antral follicle population in the sheep.

Follicles were obtained from the ovaries of four groups of 15 ewes. Ewes in the control group were ovariectomized on the 12th day of the oestrous cycle. The other ewes were all given PMSG on the 12th day of the cycle; some were ovariectomized 24 or 40 h later, the others were given prostaglandin followed by hCG and were ovariectomized 6 or 12 h after the hCG injection. All follicles greater than 2 mm in diameter were measured and examined macroscopically for signs of atresia. Some were subjected to detailed morphological examination, the pattern of steroid secretion was determined in others. All the evidence from these three approaches suggested that, in vivo, reversal of the atretic process ('rescue') plays no part in the increase in the number of follicles observed following administration of PMSG.     

Effect of follicle size on bovine oocyte quality and developmental competence following maturation,fertilization, and culture in vitro

The aim of the present series of experiments was to investigate the effect of the size of follicle from which the oocytes originate on their subsequent in vitro developmental ability. Ovarian follicles were isolated and grouped according to size (2–6 mm, >6 mm). Primary oocytes were carefully liberated and grouped according to morphology into one of five categories: denuded; expanded; with two or three layers of cumulus; with four or five layers; and with many (six or more) layers. Following in vitro maturation (IVM), fertilization (IVF), and culture (IVC), more oocytes with many layers of cumulus (P < 0.01, 70.2%, 73/104 vs. 46.8%, 87/186, respectively) and a higher proportion of blastocysts were obtained from follicles > 6 mm compared to 2–6 mm follicles (P < 0.01, 65.9%, 60/91 from >6 mm follicles vs. 34.3%, 34/99 from 2–6 mm follicles, respectively). Use of follicular fluid (BFF) from follicles of different sizes in the IVM medium did not significantly increase the cleavage rate or blastocyst yield compared to controls. Administration of procine folliclestimulating hormone (pFSH) to donors prior to slaughter was investigated as a possible means of increasing the number of larger sized follicles in the ovaries and, thereby, the quality of the recovered oocytes. It was found that administration of six injections of pFSH beginning 3 days prior to slaughter resulted in a significant increase (P < 0.001) in the proportion of follicles >6 mm in diameter (31.6%) compared to that in nontreated controls (6.6%) and to animals that received only four injection groups (9.4%). The blastocyst yield from oocytes originating from >6 mm follicles, whether from unstimulated or from pFSH-treated animals, was approximately double that of oocytes from 2–6 mm follicles (P < 0.01; 42.9%, 24/56 for >6 mm follicles vs. 22.8%, 21/92 for 2–6 mm follicles, respectively, for the 6 pFSH group; P > 0.05; 62.5%, 5/8 for >6 mm follicles vs. 32.8%, 22/67 for 2–6 mm follicles, respectively, for the control). © 1994 Wiley-Liss, Inc.     

In vitro fertilization with normal development in the sheep

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two-to six-cell stage within 40 h (75. 8% for ovulated and 62. 6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20–22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61. 9%) and 10 (66. 6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained ( > 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress ( > 3 months).     

Ovarian follicular dynamics after cauterization of the dominant follicle in anestrous ewes

An experiment was conducted to ascertain if follicles could reach ovulatory size after the largest follicle (dominant) has been removed at different times during a progestin treatment in anestrous ewes, and secondly to determine if these new follicles could respond to an hCG-induced ovulation and have similar function as corpora lutea. Mature crossbred sheep (n=44) in anestrous were treated with an intravaginal sponge containing 40 mg of FGA (day 0=sponge insertion) for 9 days. Treatments consisted of cauterization of the largest follicle on the experimental day 3 (T1), day 6 (T2) and day 9 (T3); day 12 to ascertain the size of the largest follicle in control ewes. During laparotomies, the diameters of the largest follicle (DF), and those of the second and third largest follicles (SF1 and SF2, respectively) were determined. On day 12, a second laparotomy was performed for those ewes which had their DF cauterized on days 3, 6 and 9, a fourth group was left intact and only laparotomized on day 12. At this time, the size of the new DF, SF1 and SF2 were determined. Immediately after the laparotomy on day 12, all the ewes were treated with 1000 i.u. of hCG to induce ovulation. Blood samples were collected daily from day 0 to 50 and samples were analyzed for progesterone concentrations. The size of the DF at the time of sponge removal was smaller that those observed on day 3 or 6 of sponge suggesting that follicles in ewes treated with this progestin regress and a new wave of follicular development ensues between day 6 and the time of sponge removal. The size of the DF on day 12 was also smaller in ewes that have the largest follicle removed at the time of sponge removal reflecting that these follicles had a shorter period of growth; however, the rate of growth was greater for these follicles than for follicles arising after cauterization on day 3 or 6 after sponge insertion. There were no differences among treatments, in the number of ewes that formed a corpus luteum (CL) in response to hCG. Life span of the corpora lutea did not differ among ewes having their DF removed on day 6 or 9 or those that served as controls, however, ewes that had their DF removed on day 3 developed longer lived CL in a larger proportion of animals. Average progesterone concentration during the life span of the induced corpora lutea was greater in control ewes than in any other experimental group. These observations allow us to conclude that, (a) the follicular dynamics observed in anestrous ewes treated with a progestin intravaginal sponge resembles that observed during the normal estrous cycle in the ewe; (b) the effects of progesterone on life span of the corpus luteum could not be only related to direct effects at the follicle but also involve changes in other components of the uterine-ovarian-hypothalamic axis; (c) the mechanisms controlling luteal life span seem to be different to those mechanisms controlling the function of the induced corpus luteum.     

Effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs

The present study was designed to determine the effect of the volume of medium (VM) and the number of oocytes (NOOC) during in vitro fertilization (IVF) on embryo development in pigs. Groups of 15, 30 and 50 in vitro matured oocytes were transferred to 2, 1 and 0.1 ml of modified Tris-buffered medium (mTBM) and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) in a 3 x 3 factorial experiment. A total of 2739 oocytes from four replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The efficiency of fertilization (EF: number of monospermic oocytes/total number of inseminated oocytes) and BF decreased (P<0.03) as the VM increased (EF: 45.9+/-2.2, 43.8+/-2.6 and 36.9+/-1.6% and BF: 29.4+/-2.7, 23.2+/-1.8 and 19.9+/-2.1% for VM 0.1, 1 and 2 ml, respectively). The BF, but not EF, was also affected (P<0.04) by NOOC (19.8+/-1.6, 28.1+/-2.3 and 24.6+/-2.9% for groups of 15, 30 and 50 oocytes, respectively). The effect of the interaction VM x NOOC on EF and BF was not significant. These results indicate that when 2000 spermatozoa/oocyte were used, a low volume of IVF medium (0.1 ml) and the number of oocytes during IVF (30-50) can improve the in vitro embryo production in pigs.     

Effects of hypoxanthine at low concentrations on in vitro maturation and fertilization of bovine oocytes

The effects of hypoxanthine (HX) at low concentrations on the ability of bovine oocyte to mature to metaphase 2 (M2) and to fertilize in vitro have been studied. It is proved that exogenous HX even at a low concentration, which was introduced into the HX-free and protein-free alpha-MEM medium supplemented with 1 μg/mL follicle-stimulating hormone, can significantly decrease the proportions of both the oocytes the matured to stage M2 and the penetrated and normally fertilized oocytes. It is indicated that the data may be used in order to develop commercial media for in vitro maturation of oocytes of mammalia, including humans.