Efficiency of Repeated In Vivo Oocyte and Embryo Recovery After rhFSH Treatment in Rabbits

This study aims to assess the efficiency of in vivo oocyte and embryo recovery after a recombinant human FSH (rhFSH) treatment in rabbit does. Females were distributed in two experimental groups: donor does were treated with rhFSH (superovulation group) for 3 days prior to artificial insemination (embryo recovery) or ovulation induction (oocyte recovery) and does without treatment remained as the control group. Mature oocytes or embryos were collected with the laparoscopy technique 16 h after ovulation induction (oocytes) or 72 h after artificial insemination (embryos). Up to four recoveries were performed with each doe. Recovery efficiencies differed significantly between embryos (84%) and oocytes (58%). Yet, the recovery rates for the superovulation and control groups did not differ. The rhFSH group was associated with a significant increase (p < 0.05) in the number of oocytes and embryos recovered in comparison with the control group (10.2 ± 1.0 and 14.3 ± 1.2 vs 6.0 ± 2.7 and 8.4 ± 2.3 for oocytes and embryos, respectively). Results from this study indicate that repeated in vivo oocyte and embryo recovery from rhFSH superovulated does maximizes the number of oocytes or embryos collected from the same female.     

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.     

Effect of hCG Treatment on the Oestrous and Ovulation Responses to FSH in Prepubertal Gilts

To ensure sufficient numbers of pregnant females, particularly at hotter times of the year, hormonal induction of gilt oestrus may be necessary. However, the gilt oestrus and ovulation responses to gonadotrophin treatment have often proven unpredictable. The objective of this study was to examine possible reasons for this unpredictability. Prepubertal gilts (approximately 150 days of age, n = 63) were assigned to one of three treatments: injection of 300 IU hCG (n = 15); pre-treatment with 100 mg FSH in polyvinylpyrrolidinone administered as 2 × 50 mg injections 24 h apart, followed by 600 IU eCG at 24 h after the second FSH injection (n = 23); or FSH pre-treatment as above followed by 300 IU hCG at 24 h after the second FSH injection (n = 25). To facilitate oestrus detection, gilts were exposed to a mature boar for 15 min daily for 7 days. Blood samples were obtained on the day of eCG or hCG injection and again 10 days later and gilt ovulation responses determined based on elevated progesterone concentrations. The oestrus responses by 7 days were 6.7%, 17.5% and 64.0% for gilts treated with hCG, FSH + eCG and FSH + hCG, respectively (p < 0.001). The oestrous gilt receiving hCG alone and one oestrous FSH + hCG gilt did not ovulate, all other oestrous gilts ovulated. A further two anoestrous FSH + eCG-treated gilts ovulated. These data suggest that FSH pre-treatment facilitated the development of ovarian follicles to the point where they became responsive to hCG, but had little effect on the response to eCG.     

Effects of Follicular Fluid on the Transport of Porcine Oocytes into the Oviduct at Ovulation

Contents Three experiments were conducted to determine whether follicular fluid (FF) enters the oviduct and plays any role in the transport of oocytes into the oviduct. Experiment 1: Oestrus and ovulation were synchronized in cycling gilts (n = 21) over a 15 day period of feeding Regumate^® and injections of 1000 IU pregnant mare’s serum gonadotrophin (PMSG) 24 h after the last Regumate^® feed and 500 IU human chorionic gonadotrophin (hCG) 80 h after PMSG. Ipsi-lateral aspiration of FF and salpingectomy (group 1, n = 7), aspiration of FF without salpingectomy (group 2, n = 7) or ligation of the oviduct between the ampulla and infundibulum (group 3, n = 7) was performed endoscopically prior to ovulation (34–36 h after hCG). Ipsi-lateral (group 2 and 3) and contra-lateral salpingectomy was carried out in all gilts post ovulation, 42–44 h after hCG. The oviducts were flushed with 1 ml saline and the samples as well as the aspirated FF were analysed for progesterone and estradiol by RIA methods. In group 1 both progesterone and estradiol concentrations did not differ before and after ovulation. Withdrawal of FF from the ipsi-lateral ovary by aspiration (group 2) or ligation of the oviduct (group 3) did not influence the steroid content within the oviducts. Similarly low progesterone concentrations were measured in ipsi- and contra-lateral oviducts after ovulation (group 2: 0.29 ± 0.17 versus 0.24 ± 0.35 ng/ml and group 3: 0.22 ± 0.19 versus 0.21 ± 0.22 ng/ml). The high content of progesterone of FF (269.7 ± 67.9 and 389.6 ± 226.5 ng/ml in group 1 and 2, respectively) was not reflected in the oviductal fluid. Experiment 2: In five gilts 0.06 ml ³H-progesterone (30 000 dpm) were applied via a fine 27 G injection needle into the largest three follicles of the ipsi-lateral ovary prior to ovulation (34–36 h after hCG). The oviducts were flushed following ovario-salpingectomy 42–44 h after hCG. All follicles had ovulated. The oviductal flushings and oviductal and ovarian tissue were analysed for labelled progesterone. No differences were measured in the content of ³H-progesterone of oviductal flushings and of both oviductal and ovarian tissues between the ipsi-lateral injection and contra-lateral control sides. The main part of the counts detected was within the range of background dpm values. Only 2.4% of the initial counts were recovered from fluid and tissue samples. Experiment 3: In a subsequent study FF was cautiously aspirated by endoscopy from follicles of the ipsi-lateral ovary 34–36 h after hCG (n = 12 gilts). Postovulatory (58 h after hCG), both oviducts were flushed and the oocytes were recovered. To test the influence of follicle puncture alone on the process of ovulation (n = 8 gilts), the aspiration needle alone was pricked into the follicles of the ipsi-lateral ovary, without any fluid aspiration. Despite the cautious aspiration of FF from 89 follicles, 26 oocytes were recovered together with the FF. Eighty-six postovulatory follicles were observed on the ipsi-lateral ovary. Out of 57 oocytes able to reach the oviduct, 29 oocytes were flushed from the oviduct (50.4 ± 28.1%). From the contra-lateral control oviduct 71 oocytes out of 91 ovulations (69.0 ± 33.9%) were recaptured. Puncture of follicles without aspiration did not influence ovulation compared with the control (recovery rate 68.2 and 79.6%, respectively). Results indicate (1) on the basis of the low progesterone level within the oviductal fluid that only a small amount of FF seems to reach the oviduct at ovulation, and (2) FF does not appear to be a compulsory carrier of the porcine oocyte at ovulation.     

Nuclear and cytoplasmic maturation of bovine oocytes cultured with dbc AMP, FSH and hCG

The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus.     

Effect of Lycopene on Cytoplasmic Maturation of Porcine Oocytes In Vitro

The aim of the present study was to improve cytoplasmic maturation of porcine oocytes by the addition of lycopene into in vitro maturation (IVM) media. We designed six experimental groups; IVM medium was supplemented with 10 IU/ml FSH, FSH and 10 IU/ml human chorionic gonadotrophin (hCG), or FSH and 7 μm lycopene in the first half of the IVM culture (0–22 h) followed by further culture (22–44 h) with or without hCG. The addition of lycopene into IVM media delayed the interruption of communication between an oocyte and the cumulus cells. Although meiotic competence was similar among the six groups, the glutathione level of matured oocytes was significantly higher in the lycopene‐supplemented group (9.89 pmol per oocyte) than that in other groups (7.25 and 7.81 pmol per oocyte). Fertilization rate was significantly improved in lycopene‐supplemented groups (58.3%) more than that in the group supplemented with FSH only (43.1%), whereas there were no differences in developmental competence among the groups (blastocyst rate: 20.1–29.5%). These results indicate that insufficient cytoplasmic maturation during conventional IVM resulted by disconnection of the gap junction between an oocyte and the cumulus cells in the early phase during IVM culture. We concluded that lycopene induced a prolonged sustainment of gap junctional communication between an oocyte and the cumulus cells during porcine IVM culture, which was an effective cytoplasmic maturation of porcine IVM oocytes.     

Induction of superovulation by immunoneutralization of endogenous inhibin in immature rats

The effects of passive immunoneutralization of endogenous inhibin on ovulation rate in immature rats were investigated. Efficiency of superovulation on production of fertilized oocytes was compared between the inhibin antiserum (inhibin-AS) and equine chorionic gonadotropin (eCG) protocols. Immature female Wistar strain rats were superovulated with a single injection of 100-200 microl inhibin-AS, with and without an injection of human chorionic gonadotropin (hCG). A total of 77.8% of the 26-30-day-old rats treated with a single injection of 100-200 microl inhibin-AS ovulated 72 h after treatment, while rats given normal goat serum (NGS; 200 microl) did not ovulate. At 28 days of age, all of the inhibin-AS treated rats ovulated when additional hCG treatment was given, whereas the number of ovulated oocytes was not affected. The number of ovulated oocytes in the inhibin-AS-hCG treated groups was significantly higher than that of the NGS-hCG treated group. In addition, plasma concentrations of FSH in the inhibin-AS-hCG treated group significantly increased compared with the NGS treated group. While the percentage of mated rats in the 200 microl inhibin-AS-hCG treated group was significantly lower than that of the 15 IU eCG-hCG treated group, the fertilization rate was comparable between the two groups. The number of fertilized oocytes in the 200 microl inhibin-AS-hCG treated group was significantly higher in comparison with the 15 IU eCG-hCG treated group. These results suggest that immunoneutralization of endogenous inhibin could be a reliable method for induction of superovulation to collect a large number of normally fertilized oocytes in immature rats.     

Addition of Recombinant Human Growth Hormone to In Vitro Maturation Medium of Bovine Oocytes

The aim of this study was to increase the bovine embryonic development rate, adding recombinant human growth hormone (rhGH) to maturation medium of bovine oocytes. Oocytes were matured for 24 h in TCM 199 Earle's salts and five treatments were developed: T1, 0.01 IU/ml of recombinant human follicle stimulating hormone (rhFSH); T2, 0.01 IU/ml of rhFSH + 100 ng/ml of rhGH; T3, 0.01 IU/ml of rhFSH + 1000 ng/ml of rhGH; T4, 100 ng/ml of rhGH; and T5, 1000 ng/ml of rhGH at 39 degrees C and 5% of CO(2) in air and saturated humidity. In vitro fertilization from cumulus-oocyte complexes was conducted in TALP-Fert medium (18-22 h) and spermatozoa were selected by Percoll gradient. Zygotes were incubated in SOFaaci medium in 5% of CO(2) in air, 5% of O(2) at 39 degrees C and saturated humidity for 11 days. There was no statistical difference in cleavage rate and embryo production on day 7 and day 9 among treatments. However, the hatching rate increased significantly in the T4 and T5 treatments (11.0 and 12.8%, respectively), compared with the T1 treatment (4.6%) (p < 0.05). Therefore, the rhGH addition to the oocyte maturation medium showed beneficial effects on the hatching rate of in vitro-produced bovine embryos.     

Timing of sequential changes in chromosome configurations during the 1st meiotic division of pig oocytes cultured in vitro

This study examines the timing of changes in chromosome configurations of pig oocytes derived from small antral follicles of follicular and/or inactive stage donors using modified 199 medium supplemented with gonadotropins (Follicle Stimulating Hormone (FSH), 10 IU/ml; Human Chorionic Gonadotropin (hCG), 10 IU/ml) and Glucosamine (0.539 mg/ml). Oocytes (n = 1,215) were fixed at the end of 3 hourly intervals from 0-48 hr of culture. Results were expressed as the percentage of oocytes at each stage of maturation for each time point. The germinal vesicle (GV) stage was observed for the first 17.6 hr; germinal vesicle breakdown (GVBD) stage between 17.6-26.4 hr; metaphase I (M-I) from 26.4-30.9 hr; anaphase I (A-I) ranged from 30.9-33.4 hr; telophase I (T-I) at 33.4-34.4 hr; and metaphase II (M-II) at 34.4-48 hr.     

人绒毛膜促性腺激素对母驴促排卵效果的研究

[目的]研究人绒毛膜促性腺激素（human chorionic gonadotropin,hCG）对母驴卵泡发育、排卵率、受胎率以及血清生殖激素水平的影响。[方法]选择优势卵泡直径在30~35 mm以及大于35 mm的母驴各30头,不同优势卵泡直径的母驴群体分别设置1个500 IU/头hCG处理组（n=10）、1个1 000 IU/头hCG处理组（n=10）、1个不接受hCG处理的对照组（n=10）。采用肌肉注射方法对各组母驴进行hCG处理。每隔24 h进行1次B超检查,观察各组母驴卵泡发育情况,测量卵泡直径;记录各组发生排卵的母驴数量,计算排卵率。对各组母驴进行人工输精,输精后第18天进行孕检,记录各组受胎母驴头数,计算各组受胎率。于hCG处理后0、24、48、72 h分别测定各组母驴血清中雌二醇（estradiol,E₂）和孕酮（progesterone,PROG）水平。[结果]2个群体母驴的卵泡直径随hCG注射剂量的增加而增大;优势卵泡直径大于35 mm的母驴群体中,肌肉注射hCG的2个组在处理后24 h内均出现排卵,而对照组母驴没有排卵;优势卵泡直径不同的2个母驴群体,在hCG处理48 h后排卵母驴数和排卵率与对照组相比均有所提高,其中,hCG处理后72 h,优势卵泡直径大于35 mm的母驴群体中,1 000 IU/头 hCG处理组的排卵率达到100%。2个母驴群体中,接受hCG处理的母驴,受胎率均高于对照,并且随hCG剂量的增加,受胎率有所提高;优势卵泡直径大于35 mm的母驴群体中,1 000 IU/头 hCG处理组的受胎率达到50%。2个母驴群体中,1 000 IU/头处理组在hCG处理后24 h的血清E₂浓度均较0 h时有较大幅度的提升,在0~72 h内血清PROG浓度的总体提升幅度较大。[结论]hCG处理可提高母驴的排卵率、受胎率以及血清中E₂和PROG水平,1 000 IU/头剂量的效果更好。     

Attempt to control the day of ovulation in cycling pony mares by associating a GnRH antagonist with hCG

With the objective of controlling the day of ovulation, 40 mares were assigned to a control or three treated groups: A3d, A4d, and A5d. The treated groups received antarelix (Teverelix 0.01 mg/kg, i.v., twice a day) for 3, 4, or 5 days from the day the dominant follicle (F1) reached 28 mm (=D0), and one injection of hCG (1600 IU, i.v.) on D1, D2, or D3, respectively. Control mares received one injection of hCG when F1 reached 35 mm. Plasma LH, FSH, progesterone, and total estrogens were assayed. In the A3d, A4d, and A5d groups, 9 (90%), 6 (60%), and 5 (50%) out of 10 mares, respectively, ovulated on the expected day (i.e. between 24 and 48 h after hCG injection). In the control group, 7/10 (70%) presented the typical response to hCG. For 3 mares in both the A4d and A5d groups, the dominant follicle at the time the treatment was started did not ovulate and ovulation was postponed for between 11 and 15 days after the end of treatment. In the treated mares, the LH surge was abolished, and total estrogens were depressed during the preovulatory peak but the concentrations of FSH were not modified. Endocrine parameters were not altered in postponed cycles. Fertility did not differ in treated and control cycles. These results demonstrate that in mares: (1) ovulation can be programmed on a specific day of a 3-day period, with a success rate of 67%, by a treatment associating antarelix and one injection of hCG; (2) nevertheless in 20% of cases the dominant follicle regresses and does not ovulate; (3) for these mares ovulation is postponed by approximately 2 weeks; (4) terminal growth of the preovulatory follicle only requires low circulating concentrations of LH but atresia induced by a GnRH antagonist is significant when this treatment is administrated for more than 18 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.     

hCG‐Induced Ovulation in Thoroughbred Mares Does Not Affect Corpus luteum Development and Function During Early Pregnancy

Our aim was to compare Corpus luteum (CL) development and blood plasma concentration of progesterone ([P₄]) in thoroughbred mares after spontaneous (Control: C) or human chorionic gonadotrophin (hCG)‐induced ovulation. Lactating mares (C = 12; hCG = 21) were daily teased and mated during second oestrus post‐partum. Treated mares received 2500 IU hCG i.v. at first day of behavioural oestrus when dominant follicular size was >35, ≤42 mm and mated 12–24 h after. Control mares in oestrus were mated with dominant follicular size ≥45 mm. Dominant follicle before ovulation, CL and gestational sac were measured by ultrasound and [P₄] by radioimmunoassay (RIA). Blood sampling and ultrasound CL exams were done at days 1, 2, 3, 4, 8, 12, 16, 20, 25, 30, 35, 40, 45, 60 and 90 after ovulation and gestational sac from day 12 after ovulation in pregnant (P) mares; non‐pregnant (NP) were followed until oestrus returned. Data analyses considered four subgroups: hCG‐P, hCG‐NP, C‐P and C‐NP. Preovulatory follicular size was smaller in hCG mares than in C: 39.2 ± 2.7 mm vs 51.0 ± 1.8 mm (p < 0.0001). All hCG mares ovulated 24–48 h after treatment and presented similar oestrus duration as controls. C. luteum size in P mares showed the same pattern of development through days 4–35, presenting erratic differences during initial establishment. Thus, on days 1 and 3, CL was smaller in hCG‐P (p < 0.05); while in hCG‐NP, CL size was greater than in C‐NP on day three (p = 0.03). Corpus luteum size remained stable until day 90 in hCG‐P mares, while in C‐P a transient and apparently not functional increase was detected on days 40 and 45 (p < 0.05) and the decrease from day 60 onwards, made this difference to disappear. No differences were observed in [P₄] pattern between P, or between NP subgroups, respectively. So, hCG‐induced ovulation does not affect CL development, neither [P₄] during early pregnancy. One cycle pregnancy rate tended to be lower in hCG mares while season pregnancy rates were similar to controls.     

The in vitro culture supplements and selected aspects of canine oocytes maturation

The maturation of oocytes is one of the most important steps determining their developmental competence. Due to the low percentage of oocytes of bitches that reach the MII stage, searching for reagents that may stimulate the growth and maturation of oocytes is still present in this species of mammals. The most important media supplements include gonadotropins (LH, FSH, hCG), growth factors (IGF, TGF, EGF, FGF), progesterone and follicular fluid. It is suggested that the supplement of EGF, and/or follicular cells may have an important influence on the percentage of cells that reach the MII stage. Despite plenty of research based on the improvement of bitch oocytes in vitro culture, the results obtained are still unsatisfactory. Moreover, in the long stages of canine oocytes maturation many molecular and morphological modifications (including changes in mitochondria structure and configuration in the cytoplasm) are involved. In this article, the influence of selected media supplements on the efficiency of bitch oocytes in vitro maturation was described. The molecular and morphological modifications during canine oocytes maturation were also considered in the text.     

Synchronization of ovulation using GnRH or hCG with the CO-Synch protocol in suckled beef cows.

The objectives of this study were to evaluate replacing GnRH with hCG and the effects of 48-h calf removal (CR) on pregnancy rates of cows synchronized with the CO-Synch protocol. Suckled beef cows (n = 467) at two locations were assigned to treatment by breed, age, and calving date. Treatment included either GnRH with (n = 121) or without CR (n = 117) or hCG with (n = 115) or without CR (n = 114) using the CO-Synch protocol. On d 0 and 9, cows received either hCG (2,500 IU, i.m.) or GnRH (100 microg, i.m.), and on d 7 all cows received PGF2alpha (25 mg). At one location, blood samples were collected from all cows (n = 203) on d -14, -7, 0, 7, 9, and 16. Calves were removed on d 7 and returned on d 9 (48 h) from approximately half of the cows that received GnRH or hCG. Cows that were detected in estrus between d 6 and 9 were bred approximately 12 h later and received no further injections. Cows not observed in estrus by d 9 received a second injection of either GnRH or hCG and were timed-inseminated. The AI pregnancy rates for GnRH-treated cows with or without CR and hCG-treated cows with or without CR were 46, 49, 35, and 34%, respectively (P = 0.44). Pregnancy rates of cows differed by treatment x age interaction (P = 0.07), hormone (P = 0.09), and hormone x age (P = 0.01) but not by CR (P = 0.66) or CR x age (P = 0.33). Among 2-yr-olds, pregnancy rates were higher for cows treated with hCG without CR than for cows that received GnRH with calf removal, whereas cows treated with hCG with CR and GnRH without CR were intermediate. In addition hCG-treated 2-yr-olds had higher pregnancy rates than GnRH-treated 2-yr-olds regardless of calf presence, but the reverse was true for older cows. Overall, GnRH-treated cows (48%) had a higher (P = 0.09) pregnancy rate than hCG-treated cows (34%). Among anestrous cows, GnRH and hCG were similar (P = 0.40) in their ability to induce ovulation and corpus luteum formation after the first and second injections of GnRH (31 and 76%, respectively) or hCG (39 and 61%, respectively). More (P = 0.001) hCG-treated cows exhibited short estrous cycles following timed AI. We conclude that hCG is not a suitable replacement for GnRH to synchronize ovulation with the CO-Synch protocol in multiparous cows, although further evaluation among primiparous cows is warranted using hCG with the CO-Synch protocol.     

Study on the oestrous synchronization in gilts by using progestin altrenogest and hCG: its effect on the follicular development, ovulation time and subsequent reproductive performance.

The aim of this study was to further investigate the effect of using progestin altrenogest and hCG to synchronize the oestrous cycle and its effect on follicular development, ovulation time and subsequent reproductive performance. Thirty crossbred gilts were divided into three groups. Group A (control) received a 5 ml of normal saline for 18 consecutive days by individually top-dressing. Groups B and C gilts received 20 mg (5 ml) of progestin altrenogest for 18 consecutive days by individually top-dressing. On day 3 (72 h) after withdrawal of progestin altrenogest, Group C gilts received hCG (500 IU, im). The follicular development and ovulation time were examined by transabdominal ultrasonography. Subsequent reproductive performances, i.e. number of total born per litter (NTB), number of live born per litter (NBA), number of stillbirth per litter (NSB), average piglet birth weight (ABW), lactation length (LL) and weaning to oestrous interval (WOI), were recorded. None of the gilts in Group A showed oestrus within 10 days after withdrawal of normal saline. Groups B (eight of 10) and C gilts (four of 10) came into oestrus at 5.6 +/- 0.5 and 6.5 +/- 0.6 days after withdrawal of progestin altrenogest, respectively. The ovulation time of Groups B and C gilts took placed at 25.0 +/- 4.7 and 25.0 +/- 5.0 h after standing oestrus, respectively. The pre-ovulatory follicular size (diameter) of Groups B and C gilts was 8.0 +/- 2.0 and 11.0 +/- 3.0 mm, respectively. A tendency of larger litter size (NTB) in Group B gilts was found when compared with Group A gilts. To conclude, using progestin altrenogest alone can be used to synchronize the oestrous cycle in gilts without unenthusiastic effect on the follicular development, ovulation time and subsequent reproductive performances. However, treatment of gilts with hCG at day 3 (72 h) after withdrawal of altrenogest had unenthusiastic effect on oestrus synchronization.     

培养介质、卵丘细胞和卵泡直径对猪卵母细胞体外成熟的影响

A类卵母细胞在mTCM 199、NCSU2 3和NCSU37体系中培养 4 4～ 5 2小时后 ,成熟率分别为 76 .1%、78.1%和 6 5 .2 %。前两者差异不显著 (P >0 .0 5 ) ,但显著高于后者 (P <0 .0 5 )。卵母细胞在添加eCG和hCG的NCSU2 3体系中的成熟率 (75 .6 % )明显高于添加FSH的LH和成熟率 (6 5 .2 % ) (P <0 .0 5 )。A、B、C三类卵母细胞在NCSU2 3的成熟率分别为 73.3%、6 0 .4 %和 11.0 % ,三者间差异显著 (P <0 .0 5 )。大 (ф >6mm)、中 (ф =3～ 6mm)和小 (ф <3mm)三种卵泡中的卵母细胞在NCSU2 3中培养后 ,成熟率分别为 5 6 .2 % ,78.1%和 5 1.9% ,中等卵泡中卵母胞的体外成熟率显著高于其他两组 (P <0 .0 5 )。     

Effects of induction of ovulation with GnRH or HCG on follicular and luteal blood flow in Holstein-Friesian heifers

The objective of this study was to compare the effects of gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) on follicular blood flow (FBF) during the pre-ovulatory period and on luteal blood flow (LBF) during dioestrus in Holstein-Friesian heifers. Twelve animals were randomly divided into two groups and treated with either intramuscular injection of 100 μg GnRH or intravenous (IV) injection of 5000 IU hCG on Day 0 (oestrus, 48 h after administration of PGF(2α) ) to induce ovulation. Follicular size (FS), FBF and time of ovulation were recorded with colour Doppler sonography at 0, 1, 3, 6, 12 and 24 h after GnRH and hCG treatment. Luteal size (LS) and LBF were investigated on Day 9 and 12 after ovulation. Plasma samples were taken to determine total oestrogens (E(tot) ) and progesterone (P(4) ) after each examination. Ovulation occurred between 24 and 48 h after treatment in all animals. No difference (p > 0.05) was observed in FS between the two treatment groups. Follicular blood flow was higher in the hCG group than that was in GnRH group at 1 h after treatment (p < 0.01). Total oestrogens were also higher (p < 0.01) in the hCG group than GnRH group; however, this difference was only obvious at 12 h after treatment. No difference (p > 0.05) was observed in LS, LBF or P(4) levels on Day 9 and 12 between treatment groups. In conclusion, the results suggest that induction of ovulation with hCG and GnRH has a temporary effect on FBF and oestrogen levels while no effect on the size of corpora lutea, LBF and P(4) levels was observed.     

Chronological changes of bovine follicular oocyte maturation in vitro

Chronological changes of bovine follicular cumulus-oocyte-complexesi were studied after in vitro maturation over a period of 48 h. According to their thickness and compactness of cumulus investments they were classified into 4 groups and cultured in enriched Ham’s F-10 medium with or without human chorionic gonadotrophin (hCG) and estradiolbenzoate (EB) for 0, 6, 12, 18, 21, 24, 27, 30 and 48 h. Representative samples were taken at each time interval for evaluation of nuclear maturation stages, ooplasm quality and size of the peri vitelline space (PVS). The results showed that oocyte nuclear breakdown (ONBD) required 6 to 12 h culture, and the peak of the first polar abstriction occurred at 24 h. The culture period required for ONBD and abstraction of the first polar body were related to the thickness and compactness of cumulus investments with and approximately 6 h delay in heavily compacted complexes. Ooplasm quality evaluation failed to show a clear trend, but the PVS increased in size from 0 h to 30 h and then, retracted again from 30 to 48 h. The overall maturation rate in the presence of hCG and EB was 79.1 %, and a substantial proportion (68.8 %) of nude or partially covered oocytes reached metaphase II stage. In the presence of hCG and EB no block at either metaphase I or at anaphase-telophase I was observed. In the absence of hCG and EB the percentage of oocytes reaching metaphase II was much lower (48.6%) in comparison with oocytes matured in the presence of these hormones (79.1 %). It was concluded a very high proportion of slaughterhouse oocytes could be matured in vitro and that the cumulus investments and addition of certain hormones affected the maturation rate.     

Production of Oocytes of Nile Tilapia (Oreochromis niloticus) for In Vitro Fertilization via Hormonal Treatments

Only a few studies have described hormonal treatments for induction of synchronicity and gamete collection in Nile tilapia (Oreochromis niloticus), both important for assortative matings in breeding programmes and essential for polyploidy technologies. In this study, we compared the effectiveness of carp pituitary extract (CPE), Nile tilapia pituitary extract (TPE), human chorionic gonadotropin (hCG) and gonadotropin‐releasing hormone (GnRH) protocols on the induction of spawning and egg production in Nile tilapia. Among the hormonal treatments analysed, only hCG was effective for producing viable gametes for in vitro fertilization. To verify the viability of this hormonal treatment, hCG was tested using different doses (1000, 2000, 3000, 4000 and 5000 IU/kg) in a large number of females (208 animals) from two Nile tilapia lines. The results indicated that hCG doses between 1000 and 5000 IU/kg could be used to induce final oocyte maturation in Nile tilapia with collection of stripped oocytes. This is the first study to report differential reproductive responses to hormonal treatment between tilapia lines: line 1 was more efficient at producing eggs and post‐hatching larvae after hCG induction than line 2. In conclusion, we demonstrated that the hCG protocol may be applied on a large scale to induce final oocyte maturation in Nile tilapia. The development of a protocol for in vitro fertilization in Nile tilapia may aid in breeding programmes and biotechnological assays for the development of genetically modified lines of Nile tilapia.